Drug conjugate

ABSTRACT

The invention relates to antibody-drug conjugates (ADC) that are potentiated by co-administration of the ADC with a moiety comprising covalently linked saponin. The invention also relates to antibody-oligonucleotide conjugates (AOC) that are potentiated by co-administration of the AOC with a moiety comprising covalently linked saponin. The invention also relates to ADCs and AOCs which are conjugated with a saponin via a covalent linker. The invention further relates to an effector moiety such as a toxin or an antisense oligonucleotide such as for example a BNA, conjugated with a saponin via a covalent linkage. The invention also relates to a BNA covalently conjugated with a targeting moiety such as an antibody. The invention also relates to therapeutic combinations comprising a first pharmaceutical composition comprising a conjugate of a cell-targeting moiety such as an antibody and an antisense oligonucleotide such as a BNA covalently bound thereto, and comprising a second pharmaceutical composition comprising either a free saponin, or a conjugate of a cell-targeting moiety such as an antibody with a saponin covalently linked thereto. Furthermore, the invention relates to any of these conjugates or therapeutic compositions or therapeutic combinations, for use as a medicament. The invention also relates to any of these conjugates or therapeutic compositions or therapeutic combinations, for use in a method for the treatment or the prophylaxis of a cancer. Finally, the invention relates to methods for producing any of these conjugates of the invention.

TECHNICAL FIELD

The invention relates to antibody-drug conjugates (ADC) that arepotentiated by co-administration of the ADC with a moiety comprisingcovalently linked saponin. The invention also relates toantibody-oligonucleotide conjugates (AOC) that are potentiated byco-administration of the AOC with a moiety comprising covalently linkedsaponin. The invention also relates to ADCs and AOCs which areconjugated with a saponin via a covalent linker. The invention furtherrelates to an effector moiety such as a toxin or an antisenseoligonucleotide such as for example a BNA, conjugated with a saponin viaa covalent linkage. The invention also relates to a BNA covalentlyconjugated with a targeting moiety such as an antibody. The inventionalso relates to therapeutic combinations comprising a firstpharmaceutical composition comprising a conjugate of a cell-targetingmoiety such as an antibody and an antisense oligonucleotide such as aBNA covalently bound thereto, and comprising a second pharmaceuticalcomposition comprising either a free saponin, or a conjugate of acell-targeting moiety such as an antibody with a saponin covalentlylinked thereto. Furthermore, the invention relates to any of theseconjugates or therapeutic compositions or therapeutic combinations, foruse as a medicament. The invention also relates to any of theseconjugates or therapeutic compositions or therapeutic combinations, foruse in a method for the treatment or the prophylaxis of a cancer.Finally, the invention relates to methods for producing any of theseconjugates of the invention.

BACKGROUND

Molecules with a therapeutic biological activity are in many occasionsin theory suitable for application as an effective therapeutic drug forthe treatment of a disease such as a cancer in human patients in needthereof. A typical example are small-molecule biologically activemoieties. However, many if not all potential drug-like molecules andtherapeutics currently used in the clinic suffer from at least one of aplethora of shortcomings and drawbacks. When administered to a humanbody, therapeutically active molecules may exert off-target effects, inaddition to the biologically activity directed to an aspect underlying ato-be-treated disease or health problem. Such off-target effects areundesired and bear a risk for induction of health- or evenlife-threatening side effects of the administered molecule. It is theoccurrence of such adverse events that cause many drug-like compoundsand therapeutic moieties to fail phase III clinical trials or even phaseIV clinical trials (post-market entry follow-up). Therefore, there is astrong desire to provide drug molecules such as small-moleculetherapeutics, wherein the therapeutic effect of the drug moleculeshould, e.g., (1) be highly specific for a biological factor orbiological process driving the disease, (2) be sufficiently safe, (3) besufficiently efficacious, (4) be sufficiently directed to the diseasedcell with little to no off-target activity on non-diseased cells, (5)have a sufficiently timely mode of action (e.g. the administered drugmolecule should reach the targeted site in the human patient within acertain time frame and should remain at the targeted site for a certaintime frame), and/or (6) have sufficiently long lasting therapeuticactivity in the patient's body, amongst others. Unfortunately, to date,‘ideal’ therapeutics with many or even all of the beneficialcharacteristics here above outlined, are not available to the patients,despite already long-lasting and intensive research and despite theimpressive progress made in several areas of the individually addressedencountered difficulties and drawbacks.

Chemotherapy is one of the most important therapeutic options for cancertreatment. However, it is often associated with a low therapeutic windowbecause it has no specificity towards cancer cells over dividing cellsin healthy tissue. The invention of monoclonal antibodies offered thepossibility of exploiting their specific binding properties as amechanism for the targeted delivery of cytotoxic agents to cancer cells,while sparing normal cells. This can be achieved by chemical conjugationof cytotoxic effectors (also known as payloads or warheads) toantibodies, to create antibody-drug conjugates (ADCs). Typically, verypotent payloads such as emtansine (DM1) are used which have a limitedtherapeutic index (a ratio that compares toxic dose to efficacious dose)in their unconjugated forms. The conjugation of DM1 to trastuzumab(ado-trastuzumab emtansine), also known as Kadcycla, enhances thetolerable dose of DM1 at least two-fold in monkeys. In the past fewdecades tremendous efforts and investments have been made to developtherapeutic ADCs. However, it remains challenging to bring ADCs into theclinic, despite promising preclinical data. The first ADC approved forclinical use was gemtuzumab ozogamicin (Mylotarg, CD33 targeted,Pfizer/Wyeth) for relapsed acute myelogenous leukemia (AML) in 2000.Mylotarg was however, withdrawn from the market at the request of theFederal Drug Administration (FDA) due to a number of concerns includingits safety profile. Patients treated with Mylotarg were more often foundto die than patients treated with conventional chemotherapy. Mylotargwas admitted to the market again in 2017 with a lower recommended dose,a different schedule in combination with chemotherapy or on its own, anda new patient population. To date, only five ADCs have been approved forclinical use, and meanwhile clinical development of approximatelyfifty-five ADCs has been halted. However, interest remains high andapproximately eighty ADCs are still in clinical development in nearlysix-hundred clinical trials at present.

Despite the potential to use toxic payloads that are normally nottolerated by patients, a low therapeutic index (a ratio that comparestoxic dose to efficacious dose) is a major problem accounting for thediscontinuance of many ADCs in clinical development, which can be causedby several mechanisms such as off-target toxicity on normal cells,development of resistance against the cytotoxic agents and prematurerelease of drugs in the circulation. A systematic review by the FDA ofADCs found that the toxicity profiles of most ADCs could be categorizedaccording to the payload used, but not the antibody used, suggestingthat toxicity is mostly determined by premature release of the payload.Of the approximately fifty-five ADCs that were discontinued, it isestimated that at least twenty-three were due to a poor therapeuticindex. For example, development of a trastuzumab tesirine conjugate(ADCT-502, HER-2 targeted, ADC therapeutics) was recently discontinueddue to a narrow therapeutic index, possibly due to an on-target,off-tissue effect in pulmonary tissue which expresses considerablelevels of HER2. In addition, several ADCs in phase 3 trials have beendiscontinued due to missing primary endpoint. For example, phase 3trials of a depatuxizumab mafodotin conjugate (ABT-414, EGFR targeted,AbbVie) tested in patients with newly diagnosed glioblastoma, and amirvetuximab soravtansine conjugate (IMGN853, folate receptor alpha(FRα) targeted, ImmunoGen) tested in patients with platinum-resistantovarian cancer, were recently stopped, showing no survival benefit. Itis important to note that the clinically used dose of some ADCs may notbe sufficient for its full anticancer activity. For example,ado-trastuzumab emtansine has an MTD of 3.6 mg/kg in humans. Inpreclinical models of breast cancer, ado-trastuzumab emtansine inducedtumor regression at dose levels at or above 3 mg/kg, but more potentefficacy was observed at 15 mg/kg. This suggests that at the clinicallyadministered dose, ado-trastuzumab emtansine may not exert its maximalpotential anti-tumor effect.

ADCs are mainly composed of an antibody, a cytotoxic moiety such as apayload, and a linker. Several novel strategies have been proposed andcarried out in the design and development of new ADCs to overcome theexisting problems, targeting each of the components of ADCs. Forexample, by identification and validation of adequate antigenic targetsfor the antibody component, by selecting antigens which have highexpression levels in tumor and little or no expression in normaltissues, antigens which are present on the cell surface to be accessibleto the circulating ADCs, and antigens which allows internalizing of ADCsinto the cell after binding; and alternative mechanisms of activity;design and optimize linkers which enhance the solubility and thedrug-to-antibody ratio (DAR) of ADCs and overcome resistance induced byproteins that can transport the chemotherapeutic agent out of the cells;enhance the DAR ratio by inclusion of more payloads, select and optimizeantibodies to improve antibody homogeneity and developability. Inaddition to the technological development of ADCs, new clinical andtranslational strategies are also being deployed to maximize thetherapeutic index, such as, change dosing schedules through fractionateddosing; perform biodistribution studies; include biomarkers to optimizepatient selection, to capture response signals early and monitor theduration and depth of response, and to inform combination studies.

An example of ADCs with clinical potential are those ADCs such asbrentuximab vedotin, inotuzumab ozogamicin, moxetumomab pasudotox, andpolatuzumab vedotin, which are evaluated as a treatment option forlymphoid malignancies and multiple myeloma. Polatuzumab vedotin, bindingto CD79b on (malignant) B-cells, and pinatuzumab vedotin, binding toCD22, are tested in clinical trials wherein the ADCs each were combinedwith co-administered rituximab, a monoclonal antibody binding to CD20and not provided with a payload [B. Yu and D. Liu, Antibody-drugconjugates in clinical trials for lymphoid malignancies and multiplemyeloma; Journal of Hematology & Oncology (2019) 12:94]. Combinations ofmonoclonal antibodies such as these examples are yet a further approachand attempt to arrive at the ‘magic bullet’ which combines many or evenall of the aforementioned desired characteristics of ADCs.

Meanwhile in the past few decades, nucleic acid-based therapeutics areunder development. Therapeutic nucleic acids can be based ondeoxyribonucleic acid (DNA) or ribonucleic acid (RNA), Anti-senseoligonucleotides (ASOs, AONs), and short interfering RNAs (siRNAs),MicroRNAs, and DNA and RNA aptamers, for approaches such as genetherapy, RNA interference (RNAi). Many of them share the samefundamental basis of action by inhibition of either DNA or RNAexpression, thereby preventing expression of disease-related abnormalproteins. The largest number of clinical trials is being carried out inthe field of gene therapy, with almost 2600 ongoing or completedclinical trials worldwide but with only about 4% entering phase 3. Thisis followed by clinical trials with ASOs. Similarly to ADCs, despite thelarge number of techniques being explored, therapeutic nucleic acidsshare two major issues during clinical development: delivery into cellsand off-target effects. For instance, ASOs such as peptide nucleic acid(PNA), phosphoramidate morpholino oligomer (PMO), locked nucleic acid(LNA) and bridged nucleic acid (BNA), are being investigated as anattractive strategy to inhibit specifically target genes and especiallythose genes that are difficult to target with small molecules inhibitorsor neutralizing antibodies. Currently, the efficacy of different ASOs isbeing studied in many neurodegenerative diseases such as Huntington'sdisease, Parkinson's disease, Alzheimer's disease, and amyotrophiclateral sclerosis and also in several cancer stages. The application ofASOs as potential therapeutic agents requires safe and effective methodsfor their delivery to the cytoplasm and/or nucleus of the target cellsand tissues. Although the clinical relevance of ASOs has beendemonstrated, inefficient cellular uptake, both in vitro and in vivo,limit the efficacy of ASOs and has been a barrier to therapeuticdevelopment. Cellular uptake can be <2% of the dose resulting in too lowASO concentration at the active site for an effective and sustainedoutcome. This consequently requires an increase of the administered dosewhich induces off-target effects. Most common side-effects areactivation of the complement cascade, the inhibition of the clottingcascade and toll-like receptor mediated stimulation of the immunesystem.

Chemotherapeutics are most commonly small molecules, however, theirefficacy is hampered by the severe off-target side toxicity, as well astheir poor solubility, rapid clearance and limited tumor exposure.Scaffold-small-molecule drug conjugates such as polymer-drug conjugates(PDCs) are macromolecular constructs with pharmacologically activity,which comprises one or more molecules of a small-molecule drug bound toa carrier scaffold (e.g. polyethylene glycol (PEG)).

Such conjugate principle has attracted much attention and has been underinvestigation for several decades. The majority of conjugates ofsmall-molecule drugs under pre-clinical or clinical development are foroncological indications. However, up-to-date only one drug not relatedto cancer has been approved (Movantik, a PEG oligomer conjugate ofopioid antagonist naloxone, AstraZeneca) for opioid-induced constipationin patients with chronic pain in 2014, which is a non-oncologyindication. Translating application of drug-scaffold conjugates intotreatment of human subjects provides little clinical success so far. Forexample, PK1 (N-(2-hydroxypropyl)methacrylamide (HPMA) copolymerdoxorubicin; development by Pharmacia, Pfizer) showed great anti-canceractivity in both solid tumors and leukemia in murine models, and wasunder clinical investigation for oncological indications. Despite thatit demonstrated significant reduction of nonspecific toxicity andimproved pharmacokinetics in man, improvements in anticancer efficacyturned out to be marginal in patients, and as a consequence furtherdevelopment of PK1 was discontinued.

The failure of scaffold-small-molecule drug conjugates is at leastpartially attributed to its poor accumulation at the tumor site. Forexample, while in murine models PK1 showed 45-250 times higheraccumulation in the tumor than in healthy tissues (liver, kidney, lung,spleen, and heart), accumulation in tumor was only observed in a smallsubset of patients in the clinical trial.

A potential solution to the aforementioned problems is application ofnanoparticle systems for drug delivery such as liposomes. Liposomes aresphere-shaped vesicles consisting of one or more phospholipid bilayers,which are spontaneously formed when phospholipids are dispersed inwater. The amphiphilicity characteristics of the phospholipids provideit with the properties of self-assembly, emulsifying and wettingcharacteristics, and these properties can be employed in the design ofnew drugs and new drug delivery systems. Drug encapsulated in aliposomal delivery system may convey several advantages over a directadministration of the drug, such as an improvement and control overpharmacokinetics and pharmacodynamics, tissue targeting property,decreased toxicity and enhanced drug activity. An example of suchsuccess is liposome-encapsulated form of a small molecule chemotherapyagent doxorubicin (Doxil: a pegylated liposome-encapsulated form ofdoxorubicin; Myocet: a non-pegylated liposomal doxorubicin), which havebeen approved for clinical use.

Therefore, a solution still needs to be found that allows for drugtherapies such as anti-tumor therapies, applicable for non-systemic usewhen desired, wherein the drug has for example an acceptable safetyprofile, little off-target activity, sufficient efficacy, sufficientlylow clearance rate from the patient's body, etc.

SUMMARY

For an embodiment of the present invention, it is a first goal toprovide an improved biologically active compound or compositioncomprising such improved biologically active compound.

It is one of several objectives of embodiments of the current inventionto provide a solution to the problem of non-specificity, encounteredwhen administering small-molecule therapeutically active compounds to ahuman patient in need thereof. It is one of several objectives ofembodiments of the current invention to provide a solution to theproblem of drugs with non-optimal specificity for a biological factor orbiological process driving a disease. It is one of several objectives ofembodiments of the current invention to provide a solution to theproblem of insufficient safety characteristics of current drugs, whenadministered to human patients in need thereof. It is one of severalobjectives of embodiments of the current invention to provide a solutionto the problem of current drugs being less efficacious than desired,when administered to human patients in need thereof. It is one ofseveral objectives of embodiments of the current invention to provide asolution to the problem of current drugs being not sufficiently directedto the diseased cell with little to no off-target activity onnon-diseased cells, when administered to human patients in need thereof.It is one of several objectives of embodiments of the current inventionto provide a solution to the problem that current drugs do not have asufficiently timely mode of action (e.g. the administered drug moleculeshould reach the targeted site in the human patient within a certaintime frame and should remain at the targeted site for a certain timeframe), when administered to human patients in need thereof. It is oneof several objectives of embodiments of the current invention to providea solution to the problem that current drugs have not sufficiently longlasting therapeutic activity in the patient's body, when administered tohuman patients in need thereof.

At least one of the above objectives of embodiments of the invention isachieved by providing a conjugate of the invention, preferablycomprising a cell-targeting moiety and at least one saponin, conjugatealso being suitable for use as a medicament or suitable for implicationin a pharmaceutical combination according to the invention, and suitablefor use as a semi-finished product in the manufacture of an ADC or anantibody-oligonucleotide conjugate (AOC) of the invention, according tothe invention. The therapeutic combination comprises for example aconjugate of the invention comprising covalently bound saponin and forexample comprises a second conjugate comprising an effector molecule,also referred to as an effector moiety or payload, wherein theconjugates comprise a different binding site for a different epitopeexposed on a different cell-surface molecule of a targeted cell, whereinthe different cell-surface molecules are expressed by the same targetcell and exposed on the surface of the same target cell, or wherein theconjugates comprise the same binding site for the same epitope on thesame cell-surface molecule of said targeted cell.

The present invention will be described with respect to particularembodiments but the invention is not limited thereto but only by theclaims. The embodiments of the invention described herein can operate incombination and cooperation, unless specified otherwise.

An aspect of the invention relates to a conjugate comprising orconsisting of an antibody and an antisense oligonucleotide such as anantisense BNA, covalently linked together. In FIG. 1-5, thegene-silencing activity of such a conjugate is depicted (in vivo test inan animal tumor model). Reference is also made to the Examples section.

An aspect of the invention relates to a combination of a firstcomposition comprising a conjugate comprising or consisting of anantibody and an antisense oligonucleotide such as an antisense BNA,covalently linked together, and a second composition comprising freesaponin of the invention (see Table A1, Scheme I). In FIG. 1-7A and inFIG. 1-7C, the gene-silencing activity of such a conjugate is depicted(in vitro cell-based bioassay with human tumor cells). Reference is alsomade to the Examples section.

An aspect of the invention relates to a pharmaceutical combinationcomprising or consisting of a first composition comprising a firstconjugate comprising or consisting of an antibody and an antisenseoligonucleotide such as an antisense BNA, and a second compositioncomprising a first conjugate comprising or consisting of the sameantibody and at least one saponin of the invention. In FIG. 1-5, thegene-silencing activity of such a conjugate is depicted (in vivo test inan animal tumor model). In FIG. 8-5, the gene-silencing activity of sucha conjugate is depicted (in vitro cell-based bioassay with human tumorcells). Reference is also made to the Examples section.

An aspect of the invention relates to a pharmaceutical combinationcomprising or consisting of a fourth composition comprising a fourthconjugate comprising or consisting of an antibody and an antisenseoligonucleotide such as an antisense BNA, and a fifth compositioncomprising a first conjugate comprising or consisting of a differentantibody and at least one saponin of the invention. In FIG. 10-6A and inFIG. 10-6C, the gene-silencing activity of such a conjugate is depicted(in vitro cell-based bioassay with human tumor cells). Reference is alsomade to the Examples section.

An aspect of the invention relates to a conjugate comprising orconsisting of an antisense oligonucleotide such as an antisense BNA,covalently linked to at least one saponin of the invention. In FIG. 1-3,the gene-silencing activity of such a conjugate is depicted (in vitrocell-based bioassay with human tumor cells). Reference is also made tothe Examples section.

An aspect of the invention relates to a conjugate comprising orconsisting of an antisense oligonucleotide such as an antisense BNA,covalently coupled to a polymeric scaffold such as a dendron such as aG4-dendron, wherein the polymeric scaffold is covalently conjugated withone or more saponin molecules of the invention, such as four saponinmolecules. In FIG. 1-3, the gene-silencing activity of such a conjugateis depicted (in vitro cell-based bioassay with human tumor cells).Reference is also made to the Examples section.

An aspect of the invention relates to a conjugate comprising orconsisting of an antibody such as a monoclonal antibody with specificityfor a tumor marker or tumor-cell receptor, covalently linked to at leastone antisense oligonucleotide molecule such as antisense BNA, andcovalently linked to at least one saponin molecule of the invention. InFIG. 2-4, the gene-silencing activity of such a conjugate is depicted(in vivo test in an animal tumor model). Reference is also made to theExamples section.

An aspect of the invention relates to a conjugate comprising orconsisting of an antibody such as a monoclonal antibody with specificityfor a tumor marker or tumor-cell receptor, covalently linked to at leastone antisense oligonucleotide molecule such as antisense BNA via atri-functional linker such as the linker of Scheme II, and covalentlylinked to at least one saponin molecule of the invention via the sametri-functional linker. In FIG. 1-1, the gene-silencing activity of sucha conjugate is depicted (in vivo test in an animal tumor model).Reference is also made to the Examples section.

An aspect of the invention relates to a therapeutic combinationconsisting or comprising of a eighth composition comprising a conjugatecomprising or consisting of an antibody, preferably a monoclonalantibody with specificity for a tumor marker or tumor-cell receptor,covalently linked to at least one saponin molecule of the invention,preferably via at least one linker, preferably at least one cleavablelinker, cleavable under physiological acidic conditions, and furthercomprising a ninth composition comprising an antisense oligonucleotidesuch as an antisense BNA molecule. In FIG. 6-2, the gene-silencingactivity of such a conjugate is depicted (in vivo test in an animaltumor model). In FIG. 5-2A and FIG. 5-2C, the gene-silencing activity ofsuch a conjugate is depicted (in vitro cell-based bioassay with humantumor cells). Reference is also made to the Examples section.

An aspect of the invention relates to any of the aforementionedconjugates or compositions or therapeutical combinations, for use as amedicament.

An aspect of the invention relates to any of the aforementionedconjugates or compositions or therapeutical combinations, for use in thetreatment or prophylaxis of a cancer.

An aspect of the invention relates to a therapeutic molecule withchemical structure of COMPOUND I:

A1_(m)((-L9_(w))((-L1_(q)-B1_(n))_(u)((-L2_(r)-L3_(s))(-L4_(v)-C)_(p))_(t)))_(x)  (compound I),

whereinA1 is a first ligand if B1 is a first effector moiety, or A1 is thefirst effector moiety if B1 is the first ligand;C is a saponin;m=0 or 1 if A1 is the first ligand and B1 is the first effector moiety;m=0-32 if A1 is the first effector moiety and B1 is the first ligand;n=0 or 1 if B1 is the first ligand and A1 is the first effector moiety,or if A1 is the first ligand and B1 is the first effector moiety;p=any of 1-128;L1 is at least one linker for covalently coupling two chemical groups;L2 is at least one linker for covalently coupling two chemical groups;L3 is at least one oligomeric or polymeric scaffold for covalentlycoupling two chemical groups;L4 is at least one linker for covalently coupling two chemical groups;L9 is a tri-functional linker for covalently coupling three chemicalgroups;q=0 or 1;r=0 or 1;s=0 or 1;t=0, 1 or 2 if s=0, and t=any of 0-16 if s=1;u=any of 0-32 if A1 is the first ligand and B1 is the first effectormoiety, or u=1 if A1 is the first effector moiety and B1 is the firstligand;v=0 or 1;w=1 or 0; andx=1-16.

An embodiment relates to the therapeutic combination comprising thetherapeutic molecule according to the invention and a second therapeuticmolecule with chemical structure of COMPOUND II:

A2_(a)((-L10_(i))((-L5_(d)-B2_(b))_(h)((-L6_(e)-L7_(f))(-L8_(i)-C)_(c))_(q)))_(k)  (compound II),

whereinA2 is a second ligand if B2 is a second effector moiety, or A2 is thesecond effector moiety if B2 is the second ligand;C is a saponin;a=0 or 1 if A2 is the second ligand and B2 is the second effectormoiety, or a=0-32 if A2 is the second effector moiety and B2 is thesecond ligand;b=0 or 1 if B2 is the second ligand and A2 is the second effectormoiety, or if A2 is the second ligand and B2 is the second effectormoiety;c=any of 1-128;L5 is at least one linker for covalently coupling two chemical groups;L6 is at least one linker for covalently coupling two chemical groups;L7 is at least one oligomeric or polymeric scaffold for covalentlycoupling two chemical groups;L8 is at least one linker for covalently coupling two chemical groups;L10 is a tri-functional linker for covalently coupling three chemicalgroups;d=0 or 1;e=0 or 1;f=0 or 1;g=0, 1 or 2 if f=0, and g=any of 0-16 if f=1;h=any of 0-32 if A2 is the second ligand and B2 is the second effectormoiety, or h=1 if A2 is the second effector moiety and B2 is the secondligand;i=0 or 1;j=1 or 0 and;k=1-16.

An embodiment relates to the therapeutic molecule of the invention,wherein r=0, s=0, v=0, s=0, v=0, p=0, t=0, ligand A1 is a monoclonalantibody or at least one binding fragment or -domain thereof accordingto any one of the claims 3-7, m=1, effector moiety B1 is an effectormoiety according to claim 8, preferably a BNA, either q=0, n=1 andu=2-4, or q=1, n=2-4, u=2-4 and linker L1 is the oligomeric or polymericscaffold L3 according to any one of the claims 21-30.

An embodiment is the therapeutic combination of the invention, whereinthe therapeutic molecule is the therapeutic molecule of the previousembodiment, and wherein the second ligand A2 is a monoclonal antibody orat least one binding fragment or -domain thereof according to theinvention, a=1, d=0, b=0, h=0, e=0, L7 is the scaffold according to theinvention and f=1, or L7 is absent and f=0, L8 is a linker or acleavable linker according to the invention, i=1, c=2-4 and g=2-4 if f=1and g=0 if f=0, and saponin C is a saponin according to the invention,preferably the saponin C is SO1861 and/or QS-21, with the proviso thatthe ligand A1 and the ligand A2 are the same or are different.

An aspect of the invention relates to a therapeutic combination, whereinthe therapeutic combination comprises: (a) a first pharmaceuticalcomposition comprising the therapeutic molecule with chemical structureof COMPOUND I according to the invention, the first pharmaceuticalcomposition optionally further comprising a pharmaceutically acceptableexcipient; and (b) a second pharmaceutical composition comprising thesecond therapeutic molecule with chemical structure of COMPOUND IIaccording to the invention, the second pharmaceutical compositionoptionally further comprising a pharmaceutically acceptable excipient.

An aspect of the invention relates to the first pharmaceuticalcomposition of the invention for use as a medicament.

An aspect of the invention relates to a therapeutic combination for usein the treatment or prevention of cancer in a human subject, wherein thetherapeutic combination comprises: (a) the first pharmaceuticalcomposition of the invention; and (b) the second pharmaceuticalcomposition of the invention, wherein the ligand A1 or B1 and the ligandA2 or B2 can bind to a tumor-cell epitope, preferably to a tumor-cellspecific epitope, on a tumor-cell surface molecule, preferably on atumor cell-specific surface molecule, with the proviso that thetumor-cell epitope or tumor-cell specific epitope to which the ligand A1or B1 can bind is the same as, or is different from the tumor-cellepitope or the tumor-cell specific epitope to which the ligand A2 or B2can bind.

An aspect of the invention relates to the first pharmaceuticalcomposition of the invention further comprising the second therapeuticmolecule of the invention.

An aspect of the invention relates to the first pharmaceuticalcomposition of the invention further comprising the second therapeuticmolecule of the invention, for use as a medicament.

An aspect of the invention relates to the first pharmaceuticalcomposition of the invention further comprising the second therapeuticmolecule of the invention, for use in the treatment or prevention of acancer in a human subject.

An aspect of the invention relates to any of the following ADCs andAOCs, and their semi-finished conjugates, comprising a conjugatecomprising covalently linked saponin of the invention and/or comprisinga conjugate comprising a payload or effector moiety linked to e.g. anantibody of the invention and either optionally further comprising atleast one effector molecule of the invention or optionally furthercomprising at least one saponin of the invention, respectively, or both:

Anti-EGFR antibody—saponin;Anti-EGFR antibody—triterpenoid saponin and/or a bisdesmosidictriterpene saponin belonging to the type of a 12,13-dehydrooleanane withan aldehyde function in position C-23 and optionally comprising aglucuronic acid function in a carbohydrate substituent at the C-3beta-OHgroup of the saponin;Anti-EGFR antibody—SO1861;Anti-EGFR antibody—GE1741;Anti-EGFR antibody—SA1641;Anti-EGFR antibody—Quil-A;Anti-EGFR antibody—QS-21;Anti-EGFR antibody—saponins in water soluble saponin fraction ofQuillaja saponaria;Cetuximab—saponin;Cetuximab—triterpenoid saponin and/or a bisdesmosidic triterpene saponinbelonging to the type of a 12,13-dehydrooleanane with an aldehydefunction in position C-23 and optionally comprising a glucuronic acidfunction in a carbohydrate substituent at the C-3beta-OH group of thesaponin;

Cetuximab—SO1861; Cetuximab—GE1741; Cetuximab—SA1641; Cetuximab—Quil-A;Cetuximab—QS-21;

Cetuximab—saponins in water soluble saponin fraction of Quillajasaponaria;Anti-HER2 antibody—saponin;Anti-HER2 antibody—triterpenoid saponin and/or a bisdesmosidictriterpene saponin belonging to the type of a 12,13-dehydrooleanane withan aldehyde function in position C-23 and optionally comprising aglucuronic acid function in a carbohydrate substituent at the C-3beta-OHgroup of the saponin;Anti-HER2 antibody—SO1861;Anti-HER2 antibody—GE1741;Anti-HER2 antibody—SA1641;Anti-HER2 antibody—Quil-A;Anti-HER2 antibody—QS-21;Anti-HER2 antibody—saponins in water soluble saponin fraction ofQuillaja saponaria;Trastuzumab—saponin;Trastuzumab—triterpenoid saponin and/or a bisdesmosidic triterpenesaponin belonging to the type of a 12,13-dehydrooleanane with analdehyde function in position C-23 and optionally comprising aglucuronic acid function in a carbohydrate substituent at the C-3beta-OHgroup of the saponin;

Trastuzumab—SO1861; Trastuzumab—GE1741; Trastuzumab—SA1641;Trastuzumab—Quil-A; Trastuzumab—QS-21;

Trastuzumab—saponins in water soluble saponin fraction of Quillajasaponaria;Anti-CD71 antibody—saponin;Anti-CD71 antibody—triterpenoid saponin and/or a bisdesmosidictriterpene saponin belonging to the type of a 12,13-dehydrooleanane withan aldehyde function in position C-23 and optionally comprising aglucuronic acid function in a carbohydrate substituent at the C-3beta-OHgroup of the saponin;Anti-CD71 antibody—SO1861;Anti-CD71 antibody—GE1741;Anti-CD71 antibody—SA1641;Anti-CD71 antibody—Quil-A;Anti-CD71 antibody—QS-21;Anti-CD71 antibody—saponins in water soluble saponin fraction ofQuillaja saponaria;OKT-9—saponin;OKT-9—triterpenoid saponin and/or a bisdesmosidic triterpene saponinbelonging to the type of a 12,13-dehydrooleanane with an aldehydefunction in position C-23 and optionally comprising a glucuronic acidfunction in a carbohydrate substituent at the C-3beta-OH group of thesaponin;

OKT-9—SO1861; OKT-9—GE1741; OKT-9—SA1641; OKT-9—Quil-A; OKT-9—QS-21;

OKT-9—saponins in water soluble saponin fraction of Quillaja saponaria;Anti-EGFR antibody—oligonucleotide;Anti-EGFR antibody—antisense oligonucleotide;Anti-EGFR antibody—siRNA;Anti-EGFR antibody—antisense BNA;Anti-EGFR antibody—antisense BNA(HSP27);Anti-EGFR antibody—proteinaceous toxin;Anti-EGFR antibody—ribosome inactivating protein;Anti-EGFR antibody—dianthin;Anti-EGFR antibody—saporin;Cetuximab—oligonucleotide;Cetuximab—antisense oligonucleotide;Cetuximab—siRNA;Cetuximab—antisense BNA;Cetuximab—antisense BNA(HSP27);Cetuximab—proteinaceous toxin;Cetuximab—ribosome inactivating protein;Cetuximab—dianthin;Cetuximab—saporin;Anti-HER2 antibody—oligonucleotide;Anti-HER2 antibody—antisense oligonucleotide;Anti-HER2 antibody—siRNA;Anti-HER2 antibody—antisense BNA;Anti-HER2 antibody—antisense BNA(HSP27);Anti-HER2 antibody—proteinaceous toxin;Anti-HER2 antibody—ribosome inactivating protein;Anti-HER2 antibody—dianthin;Anti-HER2 antibody—saporin;Trastuzumab—oligonucleotide;Trastuzumab—antisense oligonucleotide;Trastuzumab—siRNA;Trastuzumab—antisense BNA;Trastuzumab—antisense BNA(HSP27);Trastuzumab—proteinaceous toxin;Trastuzumab—ribosome inactivating protein;Trastuzumab—dianthin;Trastuzumab—saporin;Anti-CD71 antibody—oligonucleotide;Anti-CD71 antibody—antisense oligonucleotide;Anti-CD71 antibody—siRNA;Anti-CD71 antibody—antisense BNA;Anti-CD71 antibody—antisense BNA(HSP27);Anti-CD71 antibody—proteinaceous toxin;Anti-CD71 antibody—ribosome inactivating protein;Anti-CD71 antibody—dianthin;Anti-CD71 antibody—saporin;OKT-9—oligonucleotide;OKT-9—antisense oligonucleotide;OKT-9—siRNA;OKT-9—antisense BNA;OKT-9—antisense BNA(HSP27);OKT-9—proteinaceous toxin;OKT-9—ribosome inactivating protein;OKT-9—dianthin;OKT-9—saporin;Anti-EGFR antibody (-oligonucleotide)(-saponin), wherein theoligonucleotide is any one or more of antisense oligonucleotide, siRNA,antisense BNA, and antisense BNA(HSP27), and wherein the saponin is anyone or more of a triterpenoid saponin and/or a bisdesmosidic triterpenesaponin belonging to the type of a 12,13-dehydrooleanane with analdehyde function in position C-23 and optionally comprising aglucuronic acid function in a carbohydrate substituent at the C-3beta-OHgroup of the saponin, SO1861, GE1741, SA1641, Quil-A, QS-21, andsaponins in water soluble saponin fraction of Quillaja saponaria,wherein the anti-EGFR antibody preferably is cetuximab;Anti-EGFR antibody (-proteinaceous toxin)(-saponin), wherein theproteinaceous toxin is any one or more of a ribosome inactivatingprotein, dianthin and saporin, and wherein the saponin is any one ormore of a triterpenoid saponin and/or a bisdesmosidic triterpene saponinbelonging to the type of a 12,13-dehydrooleanane with an aldehydefunction in position C-23 and optionally comprising a glucuronic acidfunction in a carbohydrate substituent at the C-3beta-OH group of thesaponin, SO1861, GE1741, SA1641, Quil-A, QS-21, and saponins in watersoluble saponin fraction of Quillaja saponaria, wherein the anti-EGFRantibody preferably is cetuximab;Anti-HER2 antibody (-oligonucleotide)(-saponin), wherein theoligonucleotide is any one or more of antisense oligonucleotide, siRNA,antisense BNA, and antisense BNA(HSP27), and wherein the saponin is anyone or more of a triterpenoid saponin and/or a bisdesmosidic triterpenesaponin belonging to the type of a 12,13-dehydrooleanane with analdehyde function in position C-23 and optionally comprising aglucuronic acid function in a carbohydrate substituent at the C-3beta-OHgroup of the saponin, SO1861, GE1741, SA1641, Quil-A, QS-21, andsaponins in water soluble saponin fraction of Quillaja saponaria,wherein the anti-HER2 antibody preferably is trastuzumab;Anti-HER2 antibody (-proteinaceous toxin)(-saponin), wherein theproteinaceous toxin is any one or more of a ribosome inactivatingprotein, dianthin and saporin, and wherein the saponin is any one ormore of a triterpenoid saponin and/or a bisdesmosidic triterpene saponinbelonging to the type of a 12,13-dehydrooleanane with an aldehydefunction in position C-23 and optionally comprising a glucuronic acidfunction in a carbohydrate substituent at the C-3beta-OH group of thesaponin, SO1861, GE1741, SA1641, Quil-A, QS-21, and saponins in watersoluble saponin fraction of Quillaja saponaria, wherein the anti-HER2antibody preferably is trastuzumab;Anti-CD71 antibody (-oligonucleotide)(-saponin), wherein theoligonucleotide is any one or more of antisense oligonucleotide, siRNA,antisense BNA, and antisense BNA(HSP27), and wherein the saponin is anyone or more of a triterpenoid saponin and/or a bisdesmosidic triterpenesaponin belonging to the type of a 12,13-dehydrooleanane with analdehyde function in position C-23 and optionally comprising aglucuronic acid function in a carbohydrate substituent at the C-3beta-OHgroup of the saponin, SO1861, GE1741, SA1641, Quil-A, QS-21, andsaponins in water soluble saponin fraction of Quillaja saponaria,wherein the anti-CD71 antibody preferably is OKT-9; andAnti-CD71 antibody (-proteinaceous toxin)(-saponin), wherein theproteinaceous toxin is any one or more of a ribosome inactivatingprotein, dianthin and saporin, and wherein the saponin is any one ormore of a triterpenoid saponin and/or a bisdesmosidic triterpene saponinbelonging to the type of a 12,13-dehydrooleanane with an aldehydefunction in position C-23 and optionally comprising a glucuronic acidfunction in a carbohydrate substituent at the C-3beta-OH group of thesaponin, SO1861, GE1741, SA1641, Quil-A, QS-21, and saponins in watersoluble saponin fraction of Quillaja saponaria, wherein the anti-CD71antibody preferably is OKT-9.

An embodiment is the first proteinaceous molecule of the invention, thesemi-finished conjugate of the invention or the conjugate of theinvention, wherein the first binding site is selected from cetuximab,trastuzumab, OKT-9, and/or wherein the effector molecule is selectedfrom dianthin, saporin and antisense BNA(HSP27), and/or wherein thesaponin is selected from SO1861, GE1741, SA1641, Quil-A, QS-21, andsaponins in water soluble saponin fraction of Quillaja saponaria.

An embodiment is the conjugate according to the invention, wherein thefirst proteinaceous molecule is selected from cetuximab, trastuzumab,OKT-9, and/or wherein the effector molecule is selected from dianthin,saporin and antisense BNA(HSP27), and/or wherein the saponin is selectedfrom SO1861, GE1741, SA1641, Quil-A, QS-21, and saponins in watersoluble saponin fraction of Quillaja saponaria.

An aspect of the invention relates to an ADC or an AOCs or asemi-finished ADC conjugate or a semi-finished AOC conjugate comprisingan antibody-saponin conjugate of the invention and comprising at leastone effector molecule of the invention and/or comprising at least onesaponin of the invention, of Structure C:

A(-S)b(-E)c   Structure C,

wherein A is the first binding site;S is the saponin;E is the effector molecule;b=0-64, preferably 0, 1, 2, 3, 4, 8, 16, 32, 64 or any whole number orfraction therein between;c=0-8, preferably 0, 1, 2, 3, 4, 6, 8 or any whole number or fractiontherein between,wherein S is coupled to A and/or E, E is coupled to A and/or S,preferably S is coupled to A and E is coupled to A.

An embodiment is the Structure C of the invention, wherein A is ananti-EGFR antibody such as cetuximab, an anti-HER2 antibody such astrastuzumab, an anti-CD71 antibody such as OKT-9, and/or wherein S isany one or more of a saponin, a triterpenoid saponin and/or abisdesmosidic triterpene saponin belonging to the type of a12,13-dehydrooleanane with an aldehyde function in position C-23 andoptionally comprising a glucuronic acid function in a carbohydratesubstituent at the C-3beta-OH group of the saponin, SO1861, GE1741,SA1641, Quil-A, QS-21, and saponins in water soluble saponin fraction ofQuillaja saponaria, and/or wherein E is any one or more of anoligonucleotide, an antisense oligonucleotide, an siRNA, an antisenseBNA, and an antisense BNA(HSP27), and/or any one or more of aproteinaceous toxin, a ribosome inactivating protein, dianthin andsaporin.

An embodiment is the Structure C of the invention, the conjugate of theinvention or the semi-finished conjugate of the invention or the firstproteinaceous molecule of the invention, wherein the saponin, ifpresent, and/or the effector molecule, if present, is covalently coupledvia at least one linker, such as a cleavable linker, and/or via at leastone oligomeric or polymeric scaffold, such as a linker based onN-ε-maleimidocaproic acid hydrazide (EMCH) succinimidyl3-(2-pyridyldithio)propionate or 3-(2-Pyridyldithio)propionic acidN-hydroxysuccinimide ester (SPDP), and1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (HATU), and such as a scaffold based on aDendron such as a G4-Dendron or a tri-functional linker such as thetri-functional linker of Scheme II, and/or wherein at least a lysineside chain and/or a cysteine side chain of the first binding site of theantibody, preferably a monoclonal antibody or fragments or domainsthereof, is involved in the covalent bond with the saponin and/or theeffector molecule and/or the linker and/or the cleavable linker and/orthe scaffold, wherein preferably the saponin and/or the effectormolecule is covalently linked to the first binding site of the antibody,preferably a monoclonal antibody, wherein the covalent link comprises orconsists of an amide bond, a hydrazone bond, a disulphide bond.

An aspect of the invention relates to the use of any of theaforementioned conjugates of the invention or any of the semi-finishedconjugates of the invention or the antibody-saponin conjugate of theinvention, as a medicament.

An aspect of the invention relates to the use of any of the conjugatesof the invention or the semi-finished conjugates of the invention or theantibody-saponin conjugates of the invention, for use in the treatmentor prophylaxis of a cancer or an auto-immune disease.

FIG. 25 and FIG. 26 show examples of ADCs of the invention withcovalently coupled saponin(s) and OACs of the invention with covalentlycoupled saponin(s).

Of course, any and all of a, b, c, d, e, f, g, h, I, j, k, m, n, p, q,r, s, t, u, v, w and/or x have the value in accordance with eachindividual embodiment and aspect of the invention for any and all of theaforementioned aspects and embodiments according to the invention. Inaddition, (tri-functional) linkers L1, L2, L4, L5, L6, L8, L9 and/orL10, if present in a molecule or conjugate or moiety of the invention,are the (tri-functional) linkers as indicated for each and any of theaforementioned aspects and embodiments of the invention, as is readilyappreciated by the skilled person. The oligomeric or polymeric scaffoldsL3 and/or L7, if present in a molecule or conjugate or moiety of theinvention, are the oligomeric or polymeric scaffolds as indicated foreach and any of the aforementioned aspects and embodiments of theinvention, as is also readily appreciated by the skilled person.Furthermore, the first ligand A1 and the first effector moiety B1, ifpresent, and the second ligand A2 and the second effector moiety B2, ifpresent, and the first effector moiety A1 and the first ligand B1, ifpresent, and the second effector moiety A2 and the second ligand B2, ifpresent, are the selected and indicated ligands and effector moieties,as disclosed for the first, second, third, fourth, fifth, and sixthseries of embodiment and aspects of the invention, and all furtherembodiments and aspects of the invention, outlined here above. Saponin Cis any one or more of the saponins referred to and listed in any of theaforementioned aspects and embodiments of the invention, in particularone or more saponins selected from Scheme I and/or Table A1.

Definitions

The term “linker” has its regular scientific meaning, and here refers toa chemical moiety or a linear stretch of amino-acid residues complexedthrough peptide bonds, which attaches a molecule or an atom to anothermolecule, e.g. to a ligand or to an effector molecule or to a scaffold.Typically, the linker comprises a chain of atoms linked by chemicalbonds. Any linker molecule or linker technology known in the art can beused in the present disclosure. Where indicated, the linker is a linkerfor covalently binding of molecules through a chemical group on such amolecule suitable for forming a covalent linkage or bond with thelinker. The linker may be a non-cleavable linker, e.g., the linker isstable in physiological conditions. The linker may be a cleavablelinker, e.g. a linker that is cleavable, in the presence of an enzyme orat a particular pH range or value, or under physiological conditionssuch as intracellular conditions in the endosomes such as the lateendosomes and the lysosomes of mammalian cells such as human cells.Exemplary linkers that can be used in the context of the presentdisclosure includes, but is not limited to, N-ε-maleimidocaproic acidhydrazide (EMCH), succinimidyl 3-(2-pyridyldithio)propionate or3-(2-Pyridyldithio)propionic acid N-hydroxysuccinimide ester (SPDP), and1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate (HATU).

The term “tri-functional linker” has its regular scientific meaning, andhere refers to a linker which attaches three molecules via a chemicalgroup on each of the three molecules. The skilled person is able todesign such tri-functional linkers, based on the present disclosure andthe common general knowledge. Such tri-functional linker can exhibit,for instance, a maleimido group that can be used for conjugation totargeting ligands that exhibit thiol groups to perform a thiol-enereaction. In addition, the tri-functional linker could exhibit adibenzocyclooctyne (DBCO) group to perform the so-called strain-promotedalkyne-azide cycloaddition (SPAAC, click chemistry) with an azidobearing saponin. Finally, the tri-functional linker could obtain a thirdfunctional group such as a trans-cyclooctene (TCO) group to perform theso-called inverse electron demand Diels-Alder (IEDDA) reaction with atetrazine (Tz) bearing effector molecule. The skilled person willappreciate that the chemical groups of the tri-functional linker can beall three the same, or different, or the linker may comprise two of thesame chemical groups for linking a molecule to the tri-functionallinker. The formed bonds between the tri-functional linker can becovalent or non-covalent, and covalent bonds are preferred. The formedbonds between the tri-functional linker and the one or two or threebound molecules via respective chemical groups, can be cleavable(labile) bonds, such as cleavable under acidic conditions inside cellssuch as endosomes and lysosomes of mammalian cells such as human cells,or can be non-cleavable bonds. Of course, the tri-functional linker mayencompass one or two chemical groups for forming covalent bonds whilethe further two or one chemical group(s), respectively, are/is forforming a non-covalent bond. Of course, the tri-functional linker mayencompass one or two chemical groups for forming cleavable bonds whilethe further two or one chemical group(s), respectively, are/is forforming a non-cleavable bond.

The term “cleavable”, such as used in the term “cleavable linker” or“cleavable bond” has its regular scientific meaning, and here refers tobeing subject to cleavage under acidic conditions, reductive conditions,enzymatic conditions or light-induced conditions. For example, acleavable linker may be subject to cleavage under acidic conditions,preferably said cleavable linker is subject to cleavage in vivo underacidic conditions as present in endosomes and/or lysosomes of mammaliancells, preferably human cells, preferably at pH 4.0-6.5, and morepreferably at pH 5.5. As another example, a cleavable linker may besubject to cleavage by an enzyme, e.g. by cathepsin. Furthermore, anexample of a covalent bond cleavable under reductive conditions is adisulphide bond.

The terms “oligomer” and “polymer” in the context of an oligomeric orpolymeric scaffold has its regular scientific meaning. A polymer hererefers to a substance which has a molecular structure built up chieflyor completely from a large number of equal or similar units bondedtogether; an oligomer here refers to a polymer whose molecules consistof relatively few repeating units. For example, a structure comprising5-10 or less equal or similar units, may be called an oligomericstructure, whereas a structure comprising 10-50 monomeric units or moremay be called a polymeric structure, whereas a structure of 10 monomericunits may be called either oligomeric or polymeric.

The term “binding site” has its regular scientific meaning, and hererefers to a region or an epitope on a molecule, e.g. a protein, DNA orRNA, to which another molecule can bind.

The term “scaffold” has its regular scientific meaning, and here refersto an oligomeric or polymeric template or a carrier or a base (basemolecule or base structure), to which one or more molecules, e.g. ligandmolecule, effector molecule, can be covalently bound, either directly,or via a linker, such as a cleavable linker. A scaffold may have astructurally ordered formation such as a polymer, oligomer, dendrimer,dendronized polymer, or dendronized oligomer or have an assembledpolymeric structure such as a hydrogel, microgel, nanogel, stabilizedpolymeric micelle or liposome, but excludes structures that are composedof non-covalent assemblies of monomers such as cholesterol/phospholipidmixtures. A scaffold may comprise a polymeric or oligomeric structure,such as poly- or oligo(amines), e.g., polyethylenimine andpoly(amidoamine); or structures such as polyethylene glycol, poly- oroligo(esters), such as poly(lactids), poly(lactams),polylactide-co-glycolide copolymers; or poly(dextrin), poly- oroligosaccharides, such as cyclodextrin or polydextrose; or structuressuch as natural and/or artificial poly- or oligoamino acids such aspoly-lysine or a peptide or a protein, DNA oligo- or polymers,stabilized RNA polymers or PNA (peptide nucleic acid) polymers.Preferably, the polymeric or oligomeric structures are biocompatible,wherein biocompatible means that the polymeric or oligomeric structuredoes not show substantial acute or chronic toxicity in organisms and canbe either excreted as it is or fully degraded to excretable and/orphysiological compounds by the body's metabolism.

The term “ligand” has its regular scientific meaning, and here refers toany molecule or molecules which may selectively bind to a targetcell-surface molecule or target cell-surface receptor expressed attarget cells, e.g. target cancer cells or target auto-immune cells. Theligand may bind to an epitope comprised by receptors or other antigenson the target cells. Preferably, the cell-binding ligands areantibodies.

The term “antibody” as used herein is used in the broadest sense, whichmay refer to an immunoglobulin (Ig) defined as a protein belonging tothe class IgG, IgM, IgE, IgA, or IgD (or any subclass thereof), or afunctional binding fragment or binding domain of an immunoglobulin. Inthe context of the present invention, a “binding fragment” or a “bindingdomain” of an immunoglobulin is defined as antigen-binding fragment or-domain or other derivative of a parental immunoglobulin thatessentially maintains the antigen binding activity of such parentalimmunoglobulin. Functional fragments and functional domains areantibodies in the sense of the present invention even if their affinityto the antigen is lower than that of the parental immunoglobulin.“Functional fragments and -domains” in accordance with the inventioninclude, but are not limited to, F(ab′)2 fragments, Fab′ fragments, Fabfragments, scFv, dsFv, single-domain antibody (sdAb), monovalent IgG,scFv-Fc, reduced IgG (rIgG), minibody, diabodies, triabodies,tetrabodies, Fc fusion proteins, nanobodies, variable V domains such asVHH, Vh, and other types of antigen recognizing immunoglobulin fragmentsand domains. The fragments and domains may be engineered to minimize orcompletely remove the intermolecular disulphide interactions that occurbetween the CH1 and CL domains. Functional fragment and -domains offerthe advantage of greater tumor penetration because of their smallersize. In addition, the functional fragment or -domain can be more evenlydistributed throughout the tumor mass as compared to wholeimmunoglobulin.

The antibodies (immunoglobulins) of the present invention may be bi- ormultifunctional. For example, a bifunctional antibody has one arm havinga specificity for one receptor or antigen, while the other armrecognizes a different receptor or antigen. Alternatively, each arm ofthe bifunctional antibody may have specificity for a different epitopeof the same receptor or antigen of the target cell.

The antibodies (immunoglobulins) of the present invention may be, butare not limited to, polyclonal antibodies, monoclonal antibodies, humanantibodies, humanized antibodies, chimeric antibodies, resurfacedantibodies, anti-idiotypic antibodies, mouse antibodies, rat antibodies,rat/mouse hybrid antibodies, llama antibodies, llama heavy-chain onlyantibodies, heavy-chain only antibodies, and veterinary antibodies.Preferably, the antibody (immunoglobulin) of the present invention is amonoclonal antibody. The resurfaced, chimeric, humanized and fully humanantibodies are also more preferred because they are less likely to causeimmunogenicity in humans. The antibodies of the ADC of the presentinvention preferably specifically binds to an antigen expressed on thesurface of a cancer cell, an autoimmune cell, a diseased cell, anaberrant cell, while leaving any healthy cell essentially unaltered(e.g. by not binding to such normal cell, or by binding to a lesserextent in number and/or affinity to such healthy cell).

Specific antibodies that can be used for the ADCs of the presentinvention include, but are not limited to, anti-HER2 monoclonal antibodysuch as trastuzumab and pertuzumab, anti-CD20 monoclonal antibody suchas rituximab, ofatumumab, tositumomab and ibritumomab, anti-CA125monoclonal antibody such as oregovomab, anti-EpCAM (17-1A) monoclonalantibody such as edrecolomab, anti-EGFR monoclonal antibody such ascetuximab, panitumumab and nimotuzumab, anti-CD30 monoclonal antibodysuch brentuximab, anti-CD33 monoclonal antibody such as gemtuzumab andhuMy9-6, anti-vascular integrin alpha-v beta-3 monoclonal antibody suchas etaracizumab, anti-CD52 monoclonal antibody such as alemtuzumab,anti-CD22 monoclonal antibody such as epratuzumab, anti-CEA monoclonalantibody such as labetuzumab, anti-CD44v6 monoclonal antibody such asbivatuzumab, anti-FAP monoclonal antibody such as sibrotuzumab,anti-CD19 monoclonal antibody such as huB4, anti-CanAg monoclonalantibody such as huC242, anti-CD56 monoclonal antibody such huN901,anti-CD38 monoclonal antibody such as daratumumab, anti-CA6 monoclonalantibody such as DS6, anti-IGF-IR monoclonal antibody such ascixutumumab and 3B7, anti-integrin monoclonal antibody such as CNTO 95,and anti-syndecan-1 monoclonal antibody such as B-134.

Any other molecules than antibodies that bind to a cell receptor orantigen of a target cell can also be used as the cell-binding ligand forthe ligand-drug conjugates of the present invention and the ligandsprovided with covalently bound saponin according to the invention. Theseligands include, but are not limited to, proteins, polypeptides,peptides, small molecules. Examples of these non-antibody ligands areinterferons (e.g. IFN-α, IFN-β, and IFN-γ), transferrins, lectins,epidermal growth factors (EGF) and EGF-like domains, gastrin-releasingpeptides (GRP), platelet-derived growth factors (PDGF), transforminggrowth factors (TGF), vaccinia growth factor (VGF), insulin andinsulin-like growth factors (IGF, e.g. IGF-1 and IGF-2), other suitablehormones such as thyrotropin releasing hormones (TRH),melanocyte-stimulating hormones (MSH), steroid hormones (e.g. estrogenand androgen), somatostatin, lymphokines (e.g. IL-2, IL-3, IL-4, andIL-6), colony-stimulating factors (CSF, e.g. G-CSF, M-CSF and GM-CSF),bombesin, gastrin, Arg-Gly-Asp or RGD, aptamers (e.g. AS-1411, GBI-10,RNA aptamers against HIV glycoprotein), small molecules (e.g. folate,anisamide phenylboronic acid), vitamins (e.g., vitamin D), carbohydrates(e.g. hyaluronic acid, galactose).

An “effector molecule” or “effector moiety” or “payload” has its regularscientific meaning and in the context of this invention is any substancethat affects the metabolism of a cell by interaction with anintracellular effector molecule target, wherein this effector moleculetarget is any molecule or structure inside cells excluding the lumen ofcompartments and vesicles of the endocytic and recycling pathway butincluding the membranes of these compartments and vesicles. Saidstructures inside cells thus include the nucleus, mitochondria,chloroplasts, endoplasmic reticulum, Golgi apparatus, other transportvesicles, the inner part of the plasma membrane and the cytosol.

The effector molecule or -moiety is a pharmaceutically active substance,such as a toxin such as a proteinaceous toxin, a drug, a polypeptide ora polynucleotide. A pharmaceutically active substance in this inventionis an effector molecule or -moiety that is used to achieve a beneficialoutcome in an organism, preferably a vertebrate, more preferably amammal such as non-human subjects or a human being/subject. Benefitsinclude diagnosis, prognosis, treatment, cure and prevention(prophylaxis) of diseases and/or symptoms and/or health problems. Thepharmaceutically active substance may also lead to undesired andsometimes even harmful side effects (adverse events such as observedduring clinical trials). In this case, pros and cons must be weighed todecide whether the pharmaceutically active substance is suitable in theparticular case. If the effect of the pharmaceutically active substanceinside a cell is predominantly beneficial for the organism as a whole,the cell is called a target cell. If the effect inside a cell ispredominantly harmful for the organism as a whole, the cell is called anoff-target cell. In artificial systems such as cell cultures andbioreactors, target cells and off-target cells depend on the purpose andare defined by the user. Examples of effector molecules and -moietiesare a drug, a toxin, a polypeptide (such as an enzyme), a polynucleotide(including polypeptides and polynucleotides that comprise non-naturalamino acids or nucleic acids), and any combination thereof.

An effector molecule or effector moiety that is a drug may include, butnot limited to, anti-cancer agents, anti-inflammatory agents, andanti-infective (e.g., anti-fungal, antibacterial, anti-parasitic,anti-viral) agents. Preferably, the drug molecule of the presentinvention is an anti-cancer agent or an anti-auto-immune agent. Suitableanti-cancer agents include, but are not limited to, alkylating agents,antimetabolites, spindle poison plant alkaloids, cytotoxic/antitumorantibiotics, topoisomerase inhibitors, photosensitizers, and kinaseinhibitors. Also included in the definition of “anti-cancer agent” are:e.g. (i) anti-hormonal agents that act to regulate or inhibit hormoneaction on tumors such as anti-estrogens and selective estrogen receptormodulators; (ii) aromatase inhibitors that inhibit the enzyme aromatase,which regulates estrogen production in the adrenal glands; (iii)anti-androgens; (iv) protein kinase inhibitors; (v) lipid kinaseinhibitors; (vi) antisense oligonucleotides, particularly those whichinhibit expression of genes in signaling pathways implicated in aberrantcell proliferation; (vii) ribozymes such as VEGF expression inhibitorsand HER2 expression inhibitors; (viii) vaccines such as gene therapyvaccines; topoisomerase 1 inhibitors; (ix) anti-angiogenic agents; andpharmaceutically acceptable salts, acids, solvates and derivatives ofany of the above.

An effector molecule or -moiety that is a toxin may include, but is notlimited to, proteinaceous toxins (e.g. bacterial-derived toxins, andplant-derived toxins), toxins targeting tubulin filaments, toxinstargeting DNA, toxins targeting RNA. Examples of proteinaceous toxinsare saporin, dianthin, ricin, modeccin, abrin, volkensin, viscumin,shiga toxin, shiga-like toxin, pseudomonas exotoxin (PE, also known asexotoxin A), diphtheria toxin (DT), and cholera toxin. Examples oftubulin filaments-targeting toxins are maytansinoids (e.g. DM1 and DM4),auristatins (e.g. Monomethyl auristatin E (MMAE) and Monomethylauristatin F (MMAF)), toxoids, tubulysins, cryptophycins, rhizoxin.Examples of DNA-targeting toxins are calicheamicins:N-Acetyl-γ-calicheamicin, CC-1065 analogs, duocarmycins, doxorubicin,methotrexate, benzodiazepines, camptothecin analogues, andanthracyclines. Examples of DNA-targeting toxins are amanitins,spliceostatins, and thailanstatins. A toxin, as used in this invention,is defined as a pharmaceutically active substance that is able to killor inactivate a cell. Preferably, a targeted toxin is a toxin that isonly, or at least predominantly, toxic for target cells but not foroff-target cells. The net effect of the targeted toxin is preferablybeneficial for the organism as a whole.

An effector molecule or -moiety that is a polypeptide may be, e.g., apolypeptide that recover a lost function, such as for instance enzymereplacement, gene regulating functions, or a toxin. Examples ofpolypeptides as effector molecules are, e.g., Cas9; toxins (e.g.saporin, dianthin, gelonin, (de)bouganin, agrostin, ricin (toxin Achain); pokeweed antiviral protein, apoptin, diphtheria toxin,pseudomonas exotoxin) metabolic enzymes (e.g. argininosuccinate lyase,argininosuccinate synthetase), enzymes of the coagulation cascade,repairing enzymes; enzymes for cell signaling; cell cycle regulationfactors; gene regulating factors (transcription factors such as NF-κB orgene repressors such as methionine repressor).

An effector molecule or an effector moiety that is a polynucleotide may,e.g., be a polynucleotide that comprises coding information, such as agene or an open reading frame encoding a protein. It may also compriseregulatory information, e.g. promotor or regulatory element bindingregions, or sequences coding for micro RNAs. Such polynucleotide maycomprise natural and artificial nucleic acids. Artificial nucleic acidsinclude, e.g. peptide nucleic acid (PNA), Morpholino and locked nucleicacid (LNA), as well as glycol nucleic acid (GNA) and threose nucleicacid (TNA). Each of these is distinguished from naturally occurring DNAor RNA by changes to the backbone of the molecule. Examples ofnucleotides as effector molecules are, but not limited to, e.g., DNA:single stranded DNA (e.g. DNA for adenine phosphoribosyltransferase);linear double stranded DNA (e.g. clotting factor IX gene); circulardouble stranded DNA (e.g. plasmids); RNA: mRNA (e.g. TAL effectormolecule nucleases), tRNA, rRNA, siRNA, miRNA, antisense RNA; anti-senseoligonucleotides (ASOs, AONs e.g. PNA, PMO, LNA and BNA).

The term “proteinaceous”, used in e.g. “proteinaceous molecule” and“proteinaceous toxin”, are molecules and toxins comprising at least astring of amino acid residues that can be obtained as an expressionproduct from a single mRNA. Such a molecule or toxin may furthercomprise any post-translational modifications, a carbohydrate such as anN- or O-linked carbohydrate, disulphide bonds, phosphorylations,sulphatations, etc., as a result of any post-translational modification,and/or may further comprise any other modification such as thoseresulting from chemical modifications (e.g., linking of effectormoieties, saponin, scaffolds, ligands, etc., either directly to e.g. anamino-acid side chain, or via at least one linker (covalently) bound tothe molecule for chemically modifying the proteinaceous molecule, andchemically bound (covalently) to the proteinaceous molecule). The term“proteinaceous” also encompasses and includes assemblies of suchmolecules, e.g. homodimers, heterotrimers, heterohexamers or complexassemblies such as ribosomes.

The terms “specific” and “specifically”, in the context of for example“specific binding” and “receptor or molecular target specificallypresent or expressed at the surface of a tumor cell” and the like, havetheir normal scientific meaning known in the art, and here refer to e.g.a binding interaction of a first molecule with a second molecule whichoccurs with a higher affinity relative to any putative binding of thefirst molecule to a further molecule different from the second molecule,or e.g. to the expression or expression to a higher extent when e.g. thenumber of receptors or molecular targets is considered, of acell-surface receptor or molecular target on the surface of a first typeof cell such as a tumor cell, autoimmune cell, diseased cell, aberrantcell, relative to the extent of expression of the same receptor ormolecular target at a second type of cell such as a healthy cell, etc.,wherein expression at the second type of cell can be fully absent orvery low, relative to any extent of expression on the tumor cell, etc.Furthermore, the term “specific”, for example in “specific binding”, hasits normal scientific meaning known in the art, and here has the meaningof indicating a molecule that can have an interaction with anothermolecule with higher binding affinity than background interactionsbetween molecules. Similarly, the term “specificity” refers to aninteraction, for example, between two molecules or between a cell and amolecule, which has higher binding affinity than background interactionsbetween molecules. Binding molecules such as immunoglobulins bind viatheir binding site such as immunoglobulin variable regions of theimmunoglobulin, to binding sites on molecules, such as epitopes,cell-surface receptors, etc., with a higher binding affinity thanbackground interactions between molecules. In the context of theinvention, background interactions are typically interactions with anaffinity lower than a K_(D) of 10E-4 M. Similarly, “specific bindingdomains” are domains that preferentially bind to binding sites onmolecules, such as epitopes, cell-surface receptors, etc., with a higherbinding affinity than background interactions between molecules. In thecontext of the invention, “background interactions” are typicallyinteractions with an affinity lower than a K_(D) of 10E-4 M. Preferably,specific binding domains bind with an affinity higher than a K_(D) ofabout 10E-5 M.

The term “binding” is defined as interactions between molecules that canbe distinguished from background interactions.

Throughout the specification, the term “fragment” refers to an aminoacid sequence which is part of a protein domain or which builds up anintact protein domain. Binding fragments according to the invention musthave binding specificity for the respective target such as acell-surface receptor, e.g. on the surface of a diseased cell such as atumor cell.

The term “ADC” or “antibody-drug conjugate” has its regular scientificmeaning known to the skilled person, and here refers to a class ofbiopharmaceutical drugs designed as a targeted therapy for treating e.g.cancer. Unlike chemotherapy, ADCs are intended to target and kill tumorcells while sparing healthy cells. ADCs are composed of an antibodylinked to a biologically active cytotoxic (anticancer) payload or drug.ADCs combine the targeting capabilities of monoclonal antibodies withthe cancer-killing ability of cytotoxic drugs. They are designed withthe intention to discriminate between healthy cells and diseased tissuesuch as tumor cells in a tumor.

The term “Saponinum album” has its normal meaning and here refers to amixture of saponins produced by Merck KGaA (Darmstadt, Germany)containing saponins from Gypsophila paniculata and Gypsophila arostii,containing SA1657 and mainly SA1641.

The term “Quillajasaponin” has its normal meaning and here refers to thesaponin fraction of Quillaja saponaria and thus the source for all otherQS saponins, mainly containing QS-18 and QS-21.

“QS-21” or “QS21” has its regular scientific meaning and here refers toa mixture of QS-21 A-apio (˜63%), QS-21 A-xylo (˜32%), QS-21 B-apio(˜3.3%), and QS-21 B-xylo (˜1.7%).

Similarly, “QS-21A” has its regular scientific meaning and here refersto a mixture of QS-21 A-apio (˜65%) and QS-21 A-xylo (˜35%).

Similarly, “QS-21B” has its regular scientific meaning and here refersto a mixture of QS-21 B-apio (˜65%) and QS-21 B-xylo (˜35%).

The term “Quil-A” refers to a commercially available semi-purifiedextract from Quillaja saponaria and contains variable quantities of morethan 50 distinct saponins, many of which incorporate thetriterpene-trisaccharide substructure Gal-(1→2)-[Xyl-(1→3)]-GlcA- at theC-3beta-OH group found in QS-7, QS-17, QS18, and QS-21. The saponinsfound in Quil-A are listed in van Setten (1995), Table 2 [Dirk C. vanSetten, Gerrit van de Werken, Gijsbert Zomer and Gideon F. A. Kersten,Glycosyl Compositions and Structural Characteristics of the PotentialImmuno-adjuvant Active Saponins in the Quillaja saponaria Molina ExtractQuil A, RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL. 9, 660-666(1995)]. Quil-A and also Quillajasaponin are fractions of saponins fromQuillaja saponaria and both contain a large variety of differentsaponins with largely overlapping content. The two fractions differ intheir specific composition as the two fractions are gained by differentpurification procedures.

The term “QS1861” and the term “QS1862” refer to QS-7 and QS-7 api.QS1861 has a molecular mass of 1861 Dalton, QS1862 has a molecular massof 1862 Dalton. QS1862 is described in Fleck et al. (2019) in Table 1,row no. 28 [Juliane Deise Fleck, Andresa Heemann Betti, Francini Pereirada Silva, Eduardo Artur Troian, Cristina Olivaro, Fernando Ferreira andSimone Gasparin Verza, Saponins from Quillaja saponaria and Quillajabrasiliensis: Particular Chemical Characteristics and BiologicalActivities, Molecules 2019, 24, 171; doi:10.3390/molecules24010171]. Thedescribed structure is the api-variant QS1862 of QS-7. The molecularmass is 1862 Dalton as this mass is the formal mass including proton atthe glucuronic acid. At neutral pH, the molecule is deprotonated. Whenmeasuring in mass spectrometry in negative ion mode, the measured massis 1861 Dalton.

The terms first, second, third and the like in the description and inthe claims, are used for distinguishing between similar elements and notnecessarily for describing a sequential or chronological order. Theterms are interchangeable under appropriate circumstances. Theembodiments of the invention can operate in other sequences thandescribed or illustrated herein.

Furthermore, the various embodiments, although referred to as“preferred” or “e.g.” or “for example” or “in particular” are to beconstrued as exemplary manners in which the invention may be implementedrather than as limiting the scope of the invention.

The term “comprising”, used in the claims, should not be interpreted asbeing restricted to the elements or steps listed thereafter; it does notexclude other elements or steps. It needs to be interpreted asspecifying the presence of the stated features, integers, steps orcomponents as referred to, but does not preclude the presence oraddition of one or more other features, integers, steps or components,or groups thereof. Thus, the scope of the expression “a pharmaceuticalcomposition comprising A and B” should not be limited to apharmaceutical composition consisting only of components A and B, ratherwith respect to the present invention, the only enumerated components ofthe pharmaceutical composition are A and B, and further the claim shouldbe interpreted as including equivalents of those components. Similarly,the scope of the expression “a method comprising step A and step B”should not be limited to a method consisting only of steps A and B,rather with respect to the present invention, the only enumerated stepsof the method are A and B, and further the claim should be interpretedas including equivalents of those steps.

In addition, reference to a feature by the indefinite article “a” or“an” does not exclude the possibility that more than one of the featuressuch as for example a component, excipient, saponin, etc. are present,unless the context clearly requires that there is one and only one ofthe features. The indefinite article “a” or “an” thus usually means “atleast one”.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1-1: in vivo HSP27 expression in A431 xenograph ‘nude’ mouse tumormodel treated with 30 mg/kg cetuximab-Cys-(SO1861-L-trifunctionallinker-L-HSP27BNA), 25 mg/kg Cetuximab-(Lys-L-HSP27BNA)⁴ or 25 mg/kgCetuximab-(Cys-L-SO1861).

FIG. 2-1: in vitro enhanced HSP27 gene silencing in EGFR expressing A431cells by treatment with cetuximab-Cys-(SO1861-L-trifunctionallinker-L-HSP27BNA), Cetuximab-(Lys-L-HSP27BNA)⁴ orCetuximab-(Cys-L-SO1861).

FIG. 3-1: The legends and axes for Figures A, B, C and D are the same.A. cell killing activity in EGFR expressing cells (MDA-MB-468) bycetuximab, cetuxamib+10 pM cetuximab-saporin,cetuximab-Cys-(dendron(-L-SO1861)⁴)^(3,9) andcetuximab-Cys-(dendron(-L-SO1861)⁴)^(3,9)+10 pM cetuximab-saporin. B.cell killing activity in HER2 expressing cells (SK-BR-3) by trastuzumab,trastuzumab+50 pM trastuzumab-saporin,Trastuzumab-Cys-(dendron(-L-SO1861)⁴)⁴ andTrastuzumab-Cys-(dendron(-L-SO1861)⁴)⁴+50 pM trastuzumab-saporin. C.cell killing activity in EGFR expressing cells (HeLa) by cetuximab,cetuxamib+10 pM cetuximab-saporin,cetuximab-Cys-(dendron(-L-SO1861)⁴)^(3,9) andcetuximab-Cys-(dendron(-L-SO1861)⁴)^(3,9)+10 pM cetuximab-saporin. D.cell killing activity in HER2 expressing cells (JIMT-1) by trastuzumab,trastuzumab+50 pM trastuzumab-saporin,Trastuzumab-Cys-(dendron(-L-SO1861)⁴)⁴ andTrastuzumab-Cys-(dendron(-L-SO1861)⁴)⁴+50 pM trastuzu mab-saporin.

FIG. 4-1: The legends and axes for Figures A, B, C and D are the same.A. cell killing activity in EGFR⁺⁺/CD71⁺ cells (MDA-MB-468) ofcetuximab, cetuximab+10 pM CD71mab-saporin,Cetuximab-Cys-(dendron(-L-SO1861)⁴)^(3,9),Cetuximab-Cys-(dendron(-L-SO1861)⁴)^(3,9)+10 pM CD71mab-saporin,Cetuximab-Lys-(dendron(-L-SO1861)⁴)^(4,4) orCetuximab-Lys-(dendron(-L-SO1861)⁴)^(4,4)+10 pM CD71mab-saporin. B. cellkilling activity in HER2⁺⁺/CD71⁺ (SK-BR-3) cell lines of trastuzumab,trastuzumab+10 pM CD71mab-saporin,trastuzumab-Cys-(dendron(-L-SO1861)⁴)⁴,trastuzumab-Cys-(dendron(-L-SO1861)⁴)⁴+10 pM CD71mab-saporin,trastuzumab-Lys-(dendron(-L-SO1861)⁴)^(4,7) ortrastuzumab-Lys-(dendron(-L-SO1861)⁴)^(4,7)+10 pM CD71mab-saporin. C.cell killing activity in EGFR⁺/CD71⁺ cells (CaSki) of cetuximab,cetuximab+10 pM CD71 mab-saporin,Cetuximab-Cys-(dendron(-L-SO1861)⁴)^(3,9),Cetuximab-Cys-(dendron(-L-SO1861)⁴)^(3,9)+10 pM CD71mab-saporin,Cetuximab-Lys-(dendron(-L-SO1861)⁴)^(4,4) orCetuximab-Lys-(dendron(-L-SO1861)⁴)^(4,4)+10 pM CD71mab-saporin. D. cellkilling activity in HER2^(+/−)/CD71⁺ cells (JIMT-1) of trastuzumab,trastuzumab+10 pM CD71 mab-saporin,trastuzumab-Cys-(dendron(-L-SO1861)⁴)⁴,trastuzumab-Cys-(dendron(-L-SO1861)⁴)⁴+10 pM CD71mab-saporin,trastuzumab-Lys-(dendron(-L-SO1861)⁴)^(4,7) ortrastuzumab-Lys-(dendron(-L-SO1861)⁴)^(4,7)+10 pM CD71mab-saporin.

FIG. 5-1: cell killing activity in HER2 expressing cells (SK-BR-3) ofT-DM1, T-DM1+25.6 nM trastuzumab or T-DM1+5.3 nMtrastuzumab-Cys-(dendron(-L-SO1861)⁴)⁴.

FIG. 6-1: HSP27 gene silencing activity of HSP27BNA-dendron(-L-SO1861)⁴compared to the HSP27BNA alone.

FIG. 7-1: Schematic representation of release of SO1861 fromdendron(-L-SO1861)⁴ under acidic conditions.

FIG. 8-1: The legends and axes for Figures A and B are the same. A. cellkilling activity in EGFR expressing A431 cells by the ‘naked’ dendron(Dendron(NEM)⁴), Dendron(NEM)⁴+10 pM EGFdianthin, dendron(-L-SO1861)⁴ ordendron(L-SO1861)⁴+10 pM EGFdianthin. B. cell killing activity in EGFRexpressing HeLa cells by the ‘naked’ dendron (Dendron(NEM)⁴),Dendron(NEM)⁴+10 pM EGFdianthin, dendron(-L-SO1861)⁴ ordendron(L-SO1861)⁴+10 pM EGFdianthin.

FIG. 9-1: The legends and axes for Figures A, B and C are the same. A.Effect of trastuzumab on the cell viability of a range of cancer cells.B. Effect of cetuximab on the cell viability of a range of cancer cells.C. Effect of T-DM1 on the cell viability of a range of cancer cells.

FIG. 10-1: Schematic representation of the monoclonalantibody-(SO1861-scaffold-antisense BNA oligo) conjugate.

FIG. 11-1: Schematic representation of the 1-target 2-component systemusing a monoclonal antibody bound to a toxin and the same monoclonalantibody bound to a scaffold comprising saponin.

FIG. 12-1: Schematic representation of the 2-target 2-component systemusing a monoclonal antibody bound to a toxin and a different monoclonalantibody with a different target bound to a scaffold comprising asaponin.

FIG. 13-1: Schematic representation of Dendron(-L-SO1861)⁴.

FIG. 14-1: Schematic representation of Dendron(-L-SO1861)⁸.

FIG. 15-1: Schematic representation of SO1861-L-trifunctionallinker-L-HSP27BNA.

FIG. 16-1: Reaction scheme of the synthesis of theDendron(SO1861)⁴-HSP27BNA oligo conjugate.

FIG. 17-1: Model scaffold consisting of four molecular arms for saponinbinding via a Schiff base (imine) and one arm for click chemistry. Thepolymeric structure is a pentavalent polyethylene glycol-based dendrimerof the first generation.

FIG. 18-1: Cell viability of HER14 cells after treatment with apentameric dendrimer (pentrimer), the pentrimer in the presence ofSA1641, dianthin-EGF, dianthin-EFG in the presence of SA1641, thepentrimer in presence of dianthin-EGF, and the pentrimer in presence ofdianthin-EGF as well as SA1641.

FIG. 19-1: SO1861 structure with highlighted chemical groups forconjugation of endosomal escape enhancing saponins to a polymericstructure. Highlighted groups are aldehyde (black circle), carboxylicacid (dashed circle), alkene (dashed pentagon), and alcohol (dashedbox).

FIG. 20-1: A. Standard molecular structure of SO-1861-EMCH conjugate.Maleimide group is marked with a circle. B. 3D model of SO1861-EMCHconjugate. Maleimide group is marked with a circle.

FIG. 21-1: Reaction scheme for the generation of poly(SO1861) usingSO1861-EMCH as monomer, the APS/TMEDA system as polymerizationinitiator, and aminopropanethiol as radical quencher.

FIG. 22-1: Schematic representation of the DNA approach. Usage of theprinciple of DNA-origami to generate a DNA based scaffold that is ableto conjugate and release glycoside molecules. In addition, one of theDNA strands obtains a click chemistry moiety that can be used forconjugation to a targeted toxin to form a functionalized scaffold. bp:base pair.

FIG. 23-1: Schematic representation of the poly(peptide-SO1861)approach. Usage of a peptide sequence that can conjugate and releaseglycoside molecules and which can react with itself to form apoly(peptide-SO1861) construct. The poly(peptide) chain endings can befurther modified with click chemistry moieties (e.g., BCN—NHS linker)that can be used for conjugation to a toxin.

FIG. 24-1: Molecular structure of G4-dendron with protected aminogroups.

FIG. 25-1: Schematic representation of a basic scaffold with clickchemistry function to link any desired effector molecule.

FIG. 26-1: Schematic representation of a functionalized scaffold withpre-bound effector molecule and click chemistry function to link anydesired ligand. Optionally, a pH-sensitive linkage can be provided torelease the effector molecule from the scaffold after reaching theendosomes.

FIG. 1-2. Antibody-protein toxin+unconjugated SO1861 vivo study. BT474tumor bearing mice treated with various concentrations ofTrastuzumab-saporin (i.v.)+1.5 mg/kg unconjugated SO1861 (subQ injection1 hour before trastuzumab-saporin treatment).

FIG. 2-2. unconjugated saponin-mediated endosomal escape and target cellkilling enhancement. A) Cell viability analyses of HeLa cells (EGFR⁺)treated with SO1861, SO1832, SO1862 (isomer of SO1861) or SO1904 with orwithout 1.5 pM EGFdianthin B) Cell viability analyses of HeLa cells(EGFR⁺) treated with EGFdianthin and fixed concentrations of SO1861,SO1832, SO1862 (isomer of SO1861) or SO1904. C) Cell viability analysesof HeLa cells (EGFR⁺) treated with SO1861 or GE1741 with or without 1.5pM EGFdianthin. D) Cell viability analyses of HeLa cells (EGFR⁺) treatedwith various QSmix (saponin mixture from Quillaia Saponaria) with orwithout 1.5 pM EGFdianthin.

FIG. 3-2. unconjugated SO1861 versus SO1861-EMCH activity. EGFR targetedantisense BNA oligo delivery and gene silencing in cancer cells,according to the invention. A, B, C) Cell viability analyses of A431(EGFR⁺⁺), HeLa (EGFR⁺) or A2058 (EGFR⁻) cells treated with SO1861 orSO1861-EMCH with or without 1.5 pM EG dianthin. D, E) Cell viabilityanalyses of A431 (EGFR⁺⁺) or HeLa (EGFR⁺) cells treated with SO1861 orSO1861-N3 with or without 1.5 pM EGFdianthin.

FIG. 4-2. unconjugated SO1861 versus SO1861-EMCH (labile) versusSO1861-S(stable). Cell viability analyses of HeLa cells (EGFR⁺) treatedwith SO1861, SO1861-S(S=HATU, stable linker) and SO1861-EMCH (labilelinker) with or without EGFdiantin.

FIG. 5-2. EGFR targeted antisense BNA oligonucleotide delivery and genesilencing. HSP27 mRNA expression analyses of A431 (EGFR⁺⁺) and A2058(EGFR⁻) cells treated with cetuximab-(Cys-L-SO1861)³⁹ orcetuximab-(Cys-L-SO1861)³⁹ 100 nM HSP27BNA.

FIG. 6-2. Tumor targeted antisense BNA oligo nucleotide delivery andgene silencing in tumor bearing mice. Mice treated withHSP27BNA+cetuximab-(Cys-L-SO1861)^(3,9) in A431 tumor bearing micereveals efficient tumor targeted gene silencing, compared to thecontrols.

FIG. 1-3: HSP27BNA gene silencing activity of HSP27BNA, HSP27BNA-SO1861and HSP27BNA-dendron-(SO1861)⁴ in A431 cancer cell lines.

FIG. 1-4: Tumor targeted protein toxin delivery results in tumor volumereduction and tumor growth inhibition, in tumor bearing mice. A. Doseescalation (intraperitoneal, i.p.) ofcetuximab-(Cys-L-SO1861)^(3,9)(Lys-S-dianthin)² in A431 tumor bearingmice reveals tumor volume reduction, compared to the control. B. Doseescalation intraperitoneal, i.p, ofcetuximab-(Cys-L-SO1861)^(3,9)(Lys-L-dianthin)² in A431 tumor bearingmice reveals tumor growth reduction, compared to the controls. C. Doseescalation intravenous, i.v., ofcetuximab-(Cys-L-SO1861)^(3,9)(Lys-L-dianthin)² in A431 tumor bearingmice reveals tumor growth reduction, compared to the controls.

FIG. 2-4: Tumor targeted antisense BNA oligo nucleotide delivery andgene silencing in tumor bearing mice. 30 mg/kgcetuximab-(Cys-L-SO1861)^(3,9)(Lys-L-HSP27BNA)^(1,8) in A431 tumorbearing mice reveals induced efficient tumor targeted gene silencing,compared to the controls.

FIG. 3-4: Tumor targeted antisense BNA oligo nucleotide delivery andgene silencing in tumor bearing mice. 30 mg/kgcetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA)³′⁷ in A431tumor bearing mice reveals induced efficient tumor targeted genesilencing, compared to the controls.

FIG. 4-4: HER2 or EGFR targeted protein toxin delivery and cell killingin cancer cells, according to the invention. A.Trastuzumab-(Cys-L-SO1861)^(3,8)(Lys-L-dianthin)^(1,7) orTrastuzumab-(Cys-L-SO1861)^(3,8)(Lys-S-dianthin)^(1,7) treatment andcontrols on SK-BR-3 cells (HER2⁺⁺) B.Trastuzumab-(Cys-L-SO1861)^(3,8)(Lys-L-dianthin)^(1,7) orTrastuzumab-(Cys-L-SO1861)^(3,8)(Lys-S-dianthin)^(1,7) treatment andcontrols on MDA-MB-468 cells (HER2⁻). C.Cetuximab-(Cys-L-SO1861)^(3,8)(Lys-L-dianthin)^(1,7) orCetuximab-(Cys-L-SO1861)^(3,8)(Lys-S-dianthin)^(1,7) treatment andcontrols on A431 cells (EGFR⁺⁺) D.Cetuximab-(Cys-L-SO1861)^(3,8)(Lys-L-dianthin)^(1,7) orCetuximab-(Cys-L-SO1861)^(3,8)(Lys-S-dianthin)^(1,7) treatment andcontrols onA2058 cells (EGFR).

FIG. 5-4: EGFR targeted antisense BNA oligo delivery and gene silencingin cancer cells, according to the invention. A.Cetuximab-(Cys-L-SO1861)^(3,8)(Lys-L-HSP27BNA)^(1,7) treatment andcontrols on A431 cells (EGFR⁺⁺) B.Cetuximab-(Cys-L-SO1861)^(3,8)(Lys-L-HSP27BNA)^(1,7) treatment andcontrols on A2058 cells (EGFR).

FIG. 6-4: HER2 targeted antisense BNA oligo delivery and gene silencingin cancer cells, according to the invention.Trastuzumab-(Cys-L-SO1861)^(3,8)(Lys-L-HSP27BNA)^(3,5) treatment andcontrols on SK-BR-3 cells (HER2⁺⁺).

FIG. 7-4: EGFR targeted antisense BNA oligo delivery and gene silencingin cancer cells, according to the invention. A.Cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA)^(3,7) treatmentand controls on A431 cells (EGFR⁺⁺). B.Cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA)³′⁷ treatmentand controls on A2058 cells (EGFR⁻).

FIG. 8-4: Control treatments on all cell lines. Cell viability whentrastuzumab (A), cetuximab (B), T-DM1, (C) free toxins: saporin anddianthin (D) or saporin coupled to a non-cell binding IgG (D) are usedas treatment on the indicated cell lines SK-BR-3, JIMT-1, MDA-MB-468,A431, CaSki, HeLa, A2058, BT-474.

FIG. 9-4: (S)n-(L)(E) concept: mAb-(SO1861)^(n)(protein toxin)^(n).Both, SO1861 at the cysteine residues (Cys) and protein toxin (ribosomalinactivating protein) at the lysine residues are conjugated to the sameantibody (mAb) for delivery and internalization into the targetcells. 1) mAb-(Cys-L-SO1861)⁴(Lys-protein toxin)² bind to itscorresponding cell surface receptor, 2) receptor-mediated endocytosisthe conjugate occurs, 3) at low endolysosomal pH and appropriateconcentration, SO1861 becomes active to enable endolysosomal escape, 4)release of toxin into cytoplasm occurs and 5) toxin induces cell death.

FIG. 10-4: (S)n-(L)(E) concept: mAb-(SO1861)^(n)(antisense BNAoligo)^(n). Both, SO1861, at the cysteine residues (Cys) and theantisense BNA oligo nucleotide, at the lysine residues are conjugated tothe same antibody (mAb) for delivery and internalization into the targetcells. 1) mAb-(Cys-SO1861)⁴(Lys-BNAoligo)² bind to its correspondingcell surface receptor, 2) receptor-mediated endocytosis of bothconjugates occurs, 3) at low endolysosomal pH and appropriateconcentration, SO1861 becomes active to enable endolysosomal escape, 4)release of BNA oligo into cytoplasm occurs and 5) target gene silencingis induced.

FIG. 11-4: (S)n—(L)(E) concept: mAb-(SO1861-scaffold-antisense BNAoligo)n. the (SO1861-trifunctional linker-BNAoligo)^(n) is conjugated toan antibody (mAb) for delivery and internalization into the targetcells. 1) mAb-(SO1861-trifunctional linker-BNAoligo)⁴ binds to itscorresponding cell surface receptor, 2) receptor-mediated endocytosis ofboth conjugates occurs, 3) at low endolysosomal pH and appropriateconcentration, SO1861 becomes active to enable endolysosomal escape, 4)release of BNA oligo into cytoplasm occurs and 5) target gene silencingis induced.

FIG. 12-4: Antibody-SO1861 conjugation procedure. Shown is the couplingreaction of the linking of four moieties of a plant-derived saponinSO1861 to the four cysteines in the light chain of an antibody. First,the disulphide bonds in the IgG are disrupted under influence ofexposure to TCEP (Tris(2-carboxyethyl)phosphine); second, the saponinSO1861 comprising a chemical linker bound to it, is added together withtrifluoro acetic acid, and four saponin moieties are linked to the IgG.For producing cleavable ‘ready to conjugate’ saponins the aldehyde groupof SO1861 was reacted with an EMCH (ε-maleimidocaproic acid hydrazide)linker. The hydrazide group of EMCH forms an acid cleavable hydrazonebond with the aldehyde of SO1861. At the same time the EMCH linkerpresents a maleimide group that is thiol (sulfhydryl group) reactive andthus can be conjugated to thiols of the IgG, i.e. the ligand moiety.Herewith, an endosomal escape enhancing conjugate of the invention isprovided, and/or a first binding molecule of the invention is provided.

FIG. 1-5: 1T2C in vivo activity. The 1T2C combination of 50 mg/kgcetuximab-(Cys-L-SO1861)⁴+25 mg/kg cetuximab-(-L-HSP27BNA)⁴ in A431tumor bearing mice reveals strong tumor targeted gene silencing,compared to the controls.

FIG. 2-5: 1T2C in vivo activity. The 1T2C combination of 40 mg/kgtrastuzumab-(Cys-L-SO1861)⁴+0.02/0.03 mg/kg trastuzumab-saporin in a PDXtumor mouse model (high HER2 expression) shows effective tumor growthinhibition.

FIG. 3-5: 1-target 2-component. EGFR targeted cell killing in A431 cells(EGFR⁺⁺) (A, C) and CaSKi cells (EGFR⁺) (B, D) by a therapeuticcombination according to the invention. A, B)Cetuximab-(Cys-L-SO1861)^(3,7) titration+fixed concentration 10 pMcetuximab-saporin and controls on A431 (A) and CaSKi (B) cells. C, D)Cetuximab-saporin titration+fixed concentration of 75 nMcetuximab-(Cys-L-SO1861)^(3,7) and controls on A431 (C) and CaSKi (D)cells. Remark: For target receptor expression data of each cell line(determined by FACS analysis) see table 19.

FIG. 4-5: 1-target 2-component. EGFR targeted cell killing in HeLa cells(EGFR^(+/−)) (A, C) and A2058 cells (EGFR⁻) (B, D) by a therapeuticcombination according to the invention. A, B)Cetuximab-(Cys-L-SO1861)^(3,7) titration+fixed concentration 10 pMcetuximab-saporin and controls on HeLa (A) and CaSKi (B) cells. C, D)Cetuximab-saporin titration+fixed concentration of 75 nMcetuximab-(Cys-L-SO1861)^(3,7) and controls on Hela (C) and A2058 (D)cells. Remark: For target receptor expression data of each cell line(determined by FACS analysis) see table 19.

FIG. 5-5: 1-target 2-component. HER2 targeted cell killing in SKBR3cells (HER2⁺⁺) (A, B) by a therapeutic combination according to theinvention. A) Trastuzumab-(Cys-L-SO1861)⁴ titration+fixed concentration50 pM trastuzumab-saporin and controls on SKBR3 cells. B)Trastuzumab-saporin titration+fixed concentration of 2.5 nMtrastuzumab-(Cys-L-SO1861)⁴ and controls on SKBR3 cells. Remark: Fortarget receptor expression data of each cell line (determined by FACSanalysis) see table 19.

FIG. 6-5: 1-target 2-component. HER2 targeted cell killing in JIMT-1cells (HER2^(+/−)) (A, C) and MDA-MB-468 cells (HER2⁻) (B, D) by atherapeutic combination according to the invention. A, B)Trastuzumab-(Cys-L-SO1861)⁴ titration+fixed concentration of 50 pMtrastuzumab-saporin and controls on JIMT-1 (A) and MDA-MB-468 (B) cells.C, D) Trastuzumab-saporin titration+fixed concentration of 2.5 nMtrastuzumab-(Cys-L-SO1861)⁴ and controls on JIMT-1 (C) and MDA-MB-468(D) cells. Remark: For target receptor expression data of each cell line(determined by FACS analysis) see table 19.

FIG. 7-5: Chloroquine inhibits the 1-target 2-component. HER2 and EGFRtargeted cell killing in SK-BR-3 (HER2⁺⁺) and A431 cells (EGFR⁺⁺), by atherapeutic combination according to the invention+chloroquine. A)Trastuzumab-saporin titration+fixed concentration of 5 nMtrastuzumab-(Cys-L-SO1861)⁴+0.5 pM chloroquine and control on SK-BR-3cells. B) Cetuximab-saporin titration+fixed concentration of 5 nMcetuximab-(Cys-L-SO1861)^(3,8)+0.5 pM chloroquine and control on A431cells. Remark: For target receptor expression data of each cell line(determined by FACS analysis) see table 19.

FIG. 8-5: 1-target 2-component. EGFR targeted gene silencing in A431cells (EGFR⁺⁺) and A2058 cells (EGFR⁻) by a therapeutic combinationaccording to the invention. A,B) Cetuximab-(Cys-L-SO1861)^(3,8)titration+fixed concentration of 100 nM Cetuximab-(Lys-L-HSP27BNA)⁴ andcontrol on A431 cells (A) and A2058 cells (B). C, D)Cetuximab-(Lys-L-HSP27BNA)⁴ titration+fixed concentration of 77 nMCetuximab-(Cys-L-SO1861)^(3,8) and control on A431 cells (C) and A2058cells (D). Remark: For target receptor expression data of each cell line(determined by FACS analysis) see table 19.

FIG. 9-5: 2-target 2-component. A) EGFR and HER2 targeted cell killingin MDA-MB-468 cells (EGFR⁺⁺) and HeLa cells (EGFR^(+/−)) and HER2targeted cell killing in SK-BR-3 cells (HER2⁺⁺) and JIMT-1 cells(HER2^(+/−)) by a therapeutic combination according to the invention. A)Cetuximab-Cys-(dendron(-L-SO1861)⁴)³′⁹ titration+fixed concentration 10pM cetuximab-saporin and controls on MDA-MB-468 cells (A) and HeLa cells(B). C,D) Trastuzumab-Cys-(dendron(-L-SO1861)⁴)⁴ titration+fixedconcentration 50 pM trastuzumab-saporin and controls on SK-BR-3 cells(C) and JIMT-1 cells (D). Remark: For target receptor expression data ofeach cell line (determined by FACS analysis) see table 19.

FIG. 10-5: 1-target 2-component. SK-BR-3 cells (HER2^(+/−)) canefficiently be killed with the therapeutic combination according to theinvention, Tratuzumab-saporin+2.5 nM trastuzumab-(Cys-L-SO1861)⁴,however titration of T-DM1+2.5 nM trastuzumab-(Cys-L-SO1861)⁴ is noteffective at such low toxin concentrations. T-DM1 isTrastuzumab-emtansine (Kadcyla®), carrying ˜3.5 emtansine (DM1) toxinmolecules per antibody (DAR3.5). Remark: For target receptor expressiondata of each cell line (determined by FACS analysis) see table 19.

FIG. 11-5: Control treatments on all cell lines. A-D) Cell viabilitywhen trastuzumab (A), cetuximab (B), T-DM1, (C) free toxins: saporin anddianthin (D) or saporin coupled to a non-cell binding IgG (D) aretreated with the indicated cell lines SK-BR-3, JIMT-1, MDA-MB-468, A431,CaSki, HeLa, A2058, BT-474. Remark: For target receptor expression dataof each cell line (determined by FACS analysis) see table 19.

FIG. 12-5: 1-target 2-component. EGFR targeted cell killing in A431cells (EGFR⁺⁺) (A) and CaSKi cells (EGFR⁺) (B) and A2058 cells (EGFR⁻)by a therapeutic combination according to the invention. A, B, C)Cetuximab-(Cys-L-QSmix)⁴′¹ titration+fixed concentration 10 pMcetuximab-saporin or 10 pM cetuximab-dianthin and controls in A431 cells(A), CaSKi cells (B) and A2058 cells (C). QSmix is a mixture of saponinsfrom an extract Quillaja saponaria. Remark: For target receptorexpression data of each cell line (determined by FACS analysis) seetable 19.

FIG. 13-5: 1-target 2-component concept: mAb1-SO1861+mAb1-protein toxin.SO1861 and toxin (ribosomal inactivating protein) are each,independently, conjugated to an antibody (mAb1) for delivery andinternalization into target cells. 1) mAb1-SO1861 and mAb1-protein toxinbind to the cell surface receptor, 2) receptor-mediated endocytosis ofboth conjugates occurs, 3) at low endolysosomal pH and appropriateconcentration, SO1861 becomes active to enable endolysosomal escape, 4)release of toxin into cytoplasm occurs and 5) toxin induces cell death

FIG. 14-5: 1-target 2-component concept: mAb1-SO1861+mAb2-BNA oligo.SO1861 and antisense BNA oligo nucleotide are each, independently,conjugated to an antibody (mAb1) for delivery and internalization intotarget cells. 1) mAb1-SO1861 and mAb1-BNAoligo bind to the cell surfacereceptor, 2) receptor-mediated endocytosis of both conjugates occurs, 3)at low endolysosomal pH and appropriate concentration, SO1861 becomesactive to enable endolysosomal escape, 4) release of BNA oligo intocytoplasm occurs and 5) target gene silencing.

FIG. 15-5: 1-target 2-component concept:mAb1-(scaffold(-SO1861)n)n+mAb1-protein toxin. Dendron(˜SO1861)^(n) andprotein toxin (ribosomal inactivating protein) are each, independently,conjugated to an antibody (mAb1) for delivery and internalization intotarget cells. 1) mAb1-dendron(-SO1861)⁴ and mAb1-protein toxin bind tothe cell surface receptor, 2) receptor-mediated endocytosis of bothconjugates occurs, 3) at low endolysosomal pH and appropriateconcentration, SO1861 becomes active to enable endolysosomal escape, 4)release of toxin into cytoplasm occurs and 5) toxin induces cell death.

FIG. 1-6: The 2T2 component system tested in A431 tumor bearing micemodel reveals tumor regression.

FIG. 2-6: The 2T2 component system tested in A431 tumor bearing micemodel reveals tumor regression and eradication.

FIG. 3-6: 2-target 2-component. EGFR/HER2 targeted cell killing in A431cells (EGFR⁺⁺/HER2^(+/−)) (A, C) and CaSKi cells (EGFR⁺⁺/HER2^(+/−)) (B,D) by a therapeutic combination according to the invention. A, B)Cetuximab-(Cys-L-SO1861)^(3,7) titration+fixed concentration 50 pMtrastuzumab-saporin and controls on A431 cells. C, D)Trastuzumab-saporin titration+fixed concentration of 75 nMcetuximab-(Cys-L-SO1861)^(3,7) and controls on Caski cells. The legendsand/or axes are the same for all the A,B, C or D.

FIG. 4-6: 2-target 2-component. EGFR/HER2 targeted cell killing in HeLacells (EGFR^(+/−)/HER2^(+/−)) (A, C) and A2058 cells (EGFR/HER2^(+/−))(B, D) by a therapeutic combination according to the invention. A, B)Cetuximab-(Cys-L-SO1861)^(3,7) titration+fixed concentration 50 pMtrastuzumab-saporin and controls on HeLa cells. C, D)Trastuzumab-saporin titration+fixed concentration of 75 nMcetuximab-(Cys-L-SO1861)^(3,7) and controls on A2058 cells. The legendsand/or axes are the same for all the A,B, C or D.

FIG. 5-6: 2-target 2-component. HER2/EGFR targeted cell killing in SKBR3cells (HER2⁺⁺/EGFR^(+/−)) (A, B) by a therapeutic combination accordingto the invention. A Trastuzumab-(Cys-L-SO1861)⁴ titration+fixedconcentration 1.5 pM EGFdianthin and controls on SKBR3 cells. B)EGFdianthin titration+fixed concentration of 2.5 nMtrastuzumab-(Cys-L-SO1861)⁴ and controls on SKBR3 cells.

FIG. 6-6: 2-target 2-component. HER2/EGFR targeted cell killing inJIMT-1 cells (HER2^(+/−)EGFR^(+/−)) (A, C) and MDA-MB-468 cells(HER2⁻/EGFR⁺⁺) (B, D) by a therapeutic combination according to theinvention. A, B) Trastuzumab-(Cys-L-SO1861)⁴ titration+fixedconcentration 1.5 pM EGFdianthin and controls on JIMT-1 cells. C, D)EGFdianthin titration+fixed concentration of 2.5 nMtrastuzumab-(Cys-L-SO1861)⁴ and controls on MDA-MB-468 cells. Thelegends and/or axes are the same for all the A,B, C or D.

FIG. 7-6: 2-target 2-component. HER2/EGFR targeted cell killing in SKBR3cells (HER2⁺⁺/EGFR^(+/−)) (A, B) by a therapeutic combination accordingto the invention. A) Trastuzumab-(Cys-L-SO1861)⁴ titration+fixedconcentration 10 pM cetuximab-saporin and controls on SKBR3 cells. B)Cetuximab-saporin titration+fixed concentration of 2.5 nMtrastuzumab-(Cys-L-SO1861)⁴ and controls on SKBR3 cells.

FIG. 8-6: 2-target 2-component. HER2/EGFR targeted cell killing inJIMT-1 cells (HER2^(+/−)EGFR^(+/−)) (A, C) and MDA-MB-468 cells(HER2⁻/EGFR⁺⁺) (B, D) by a therapeutic combination according to theinvention. A, B) Trastuzumab-(Cys-L-SO1861)⁴ titration+fixedconcentration 10 pM cetuximab-saporin and controls on JIMT-1 cells. C,D) Cetuximab-saporin titration+fixed concentration of 2.5 nMtrastuzumab-(Cys-L-SO1861)⁴ and controls on MDA-MB-468 cells. Thelegends and/or axes are the same for all the A,B, C or D.

FIG. 9-6: Chloroquine inhibits the 2-target 2-component. EGFR/HER2,EGFR/CD71 or HER2/CD71 targeted cell killing in A431 cells(EGFR⁺⁺/HER2^(+/−)/CD71⁺) (A, B), MDA-MB-468 cells (EGFR⁺⁺/HER2⁻/CD71⁺)(C) or SK-BR-3 (HER2⁺⁺/EGFR^(+/−)/CD71⁺) (D) by a therapeuticcombination according to the invention+chloroquine. A)Trastuzumab-dianthin or trastuzumab-saporin titration+fixedconcentration of 75 nM cetuximab-(Cys-L-SO1861)^(3,9)+800 nM chloroquineand controls on A431 cells. B) CD71mab-saporin titration+fixedconcentration of 10.5 nM cetuximab-(Cys-L-SO1861)^(3,9)+500 nMchloroquine and control on A431 cells. C) CD71mab-saporintitration+fixed concentration of 10.5 nMcetuximab-(Cys-L-SO1861)^(3,9)+500 nM chloroquine and control onMDA-MB-468 cells. D) CD71mab-saporin titration+fixed concentration of 5nM trastuzumab-(Cys-L-SO1861)³′⁹+500 nM chloroquine and control onSK-BR-3 cells.

FIG. 10-6: 2-target 2-component. EGFR/HER2 targeted gene silencing inA431 cells (EGFR⁺⁺/HER2^(+/−)) (A) and A2058 cells (EGFR/HER2^(+/−)) (B)by a therapeutic combination according to the invention. A)Cetuximab-(Cys-L-SO1861)^(3,9) titration+fixed concentration of 100 nMtrastuzumab-(Lys-L-HSP27BNA)^(4,4) and control on A431 cells (A) andA2058 cells (B). C, D) Trastuzumab-(Lys-L-HSP27BNA)^(4,4)titration+fixed concentration of 77 nM cetuximab-(Cys-L-SO1861)^(3,9)and controls on A431 cells (A) and A2058 cells (B). The legends and/oraxes are the same for all the A,B, C or D.

FIG. 11-6: 2-target 2-component. A) EGFR/CD71 or HER2/CD71 targeted cellkilling in MDA-MB-468 cells (EGFR⁺⁺/CD71⁺) (A) HeLa cells(EGFR^(+/−)/CD71⁺), SK-BR-3 cells (HER2⁺⁺/CD71⁺) (B) and JIMT-1 cells(HER2^(+/−)/CD71⁺) by a therapeutic combination according to theinvention. A) Cetuximab-Cys-(dendron(-L-SO1861)⁴)^(3,9) titration+fixedconcentration 10 pM CD71 mab-saporin and controls on MDA-MB-468 cells.B) A) Cetuximab-Cys-(dendron(-L-SO1861)⁴)^(3,9) titration+fixedconcentration 10 pM CD71mab-saporin and controls on HeLa cells. C)Trastuzumab-Cys-(dendron(-L-SO1861)⁴)⁴ titration+fixed concentration 10pM CD71mab-saporin and controls on SK-BR-3 cells. D)Trastuzumab-Cys-(dendron(-L-SO1861)⁴)⁴ titration+fixed concentration 10pM CD71mab-saporin and controls on JIMT-1 cells.

FIG. 12-6: 2-target 2-component versus T-DM1. A431 cells(EGFR⁺⁺/HER2^(+/−)) can efficiently be killed with the therapeuticcombination according to the invention, Tratuzumab-saporin+75 nMcetuximab-(Cys-L-SO1861)^(3,9), however titration of T-DM1+75 nMcetuximab-(Cys-L-SO1861)^(3,9) is not effective at such low toxinconcentrations. T-DM1 is Trastuzumab-emtansine (Kadcyla®), carrying=3.5emtansine (DM1) toxin molecules per antibody.

FIG. 13-6: 2-target 2-component. EGFR/CD71 and EGFR/HER2 targeted cellkilling in A431 cells (EGFR+⁺⁺/HER2^(+/−)) (A) and CaSKi cells(EGFR⁺⁺/HER2^(+/−)) (B) and A2058 cells (EGFR/HER2^(+/−)) by atherapeutic combination according to the invention. A, B,C)Cetuximab-(Cys-L-QSmix)⁴′¹ titration+fixed concentration 10 pMtrastuzumab-saporin or 10 pM CD71mab-saporin and controls on A431 cells(A). CaSKi cells (B) and A2058 cells (C). QSmix is a mixture of saponinsfrom an extract Quillaja saponaria.

FIG. 14-6: Control treatments on all cell lines. A-D) Cell viabilitywhen trastuzumab (A), cetuximab (B), T-DM1, (C) free toxins: saporin anddianthin (D) or saporin coupled to a non-cell binding IgG (D) aretreated with the indicated cell lines SK-BR-3, JIMT-1, MDA-MB-468, A431,CaSki, HeLa, A2058, BT-474. The legends and/or axes are the same for allthe A,B, C or D.

FIG. 15-6: 2-target 2-component concept: mAb1-SO1861+mAb2-protein toxin.SO1861 and toxin (ribosomal inactivating protein) are each, separately,conjugated to an antibody (mAb) for delivery and internalization intotarget cells. 1) mAb1-SO1861 and mAb2-protein toxin bind to theircorresponding cell surface receptor, 2) receptor-mediated endocytosis ofboth conjugates occurs, 3) at low endolysosomal pH and appropriateconcentration, SO1861 becomes active to enable endolysosomal escape, 4)release of toxin into cytoplasm occurs and 5) toxin induces cell death.

FIG. 16-6: 2-target 2-component concept: mAb1-SO1861+mAb2-BNA oligo.SO1861 and antisense BNA oligo nucleotide are each, separately,conjugated to an antibody (mAb) for delivery and internalization intotarget cells. 1) mAb1-SO1861 and mAb2-BNAoligo bind to theircorresponding cell surface receptor, 2) receptor-mediated endocytosis ofboth conjugates occurs, 3) at low endolysosomal pH and appropriateconcentration, SO1861 becomes active to enable endolysosomal escape, 4)release of BNA oligo into cytoplasm occurs and 5) target gene silencing.

FIG. 1-7: EGFR targeted gene silencing in A431 cells (EGFR⁺⁺) (A) andA2058 cells (EGFR⁻) (B) by a combination with unconjugated SO1861-EMCHand HSP27BNA, and SO1861-EMCH and Cetuximab-(Lys-L-HSP27BNA)titration+fixed concentration of 4000 nM SO1861-EMCH and controls onA431 cells (A) and A2058 cells (B). (A) and (B) are HSP27BNA orCetuximab-HSP27BNA conjugate (nM) based graphs. EGFR targeted genesilencing in A431 cells (EGFR⁺⁺) (A) and A2058 cells (EGFR⁻) (B) by acombination with unconjugated SO1861-EMCH with free HSP27BNA, andSO1861-EMCH and conjugate Cetuximab-(Lys-L-HSP27BNA) titration+fixedconcentration of 4000 nM SO1861-EMCH and controls on A431 cells (C) andA2058 cells (D). (C) and (D) are HSP27BNA (nM) based graphs. The legendwith FIGS. 1-7B and D also applies for FIGS. 1-7A and C.

DETAILED DESCRIPTION

In order for a bioactive molecule to work, the molecule must be able toengage with its target, e.g. in the blood serum, on the outside of thecell surface or inside a cell or an organelle. The active moiety ofalmost all protein-based targeted toxins, e.g., must enter the cytosolof the target cell to mediate its target modulatory effect. In manyconstellations the toxin remains ineffective since (1) the targetingmoiety is poorly internalized and remains bound to the outside of thecells, (2) is recycled back to the cell surface after internalization or(3) transported to the endolysosomes where it is degraded. Althoughthese fundamental issues are known for decades and more than 500targeted toxins have been investigated in the past decades, the problemshave not been solved yet and only one antibody-targeted protein toxin,moxetumomab pasudotox-tdfk (LUMOXITI®, AstraZeneca Pharmaceuticals LP),has been approved for relapsed or refractory hairy cell leukemia by theFDA to date.

To overcome these problems, many strategies have been describedincluding approaches to redirect the toxins to endogenous cellularmembrane transport complexes of the biosynthetic pathway in theendoplasmic reticulum and techniques to disrupt or weaken the membraneintegrity of endosomes, i.e. the compartments of the endocytic pathwayin a cell, and thus facilitating the endosomal escape. This comprisesthe use of lysosomotropic amines, carboxylic ionophores, calcium channelantagonists, various cell-penetrating peptides of viral, bacterial,plant, animal, human and synthetic origin, other organic molecules andlight-induced techniques. Although the efficacy of the targeted toxinswas typically augmented in cell culture hundred- or thousand-fold, inexceptional cases more than million-fold, the requirement toco-administer endosomal escape enhancers with other substances harborsnew problems including additional side effects, loss of targetspecificity, difficulties to determine the therapeutic window and celltype-dependent variations.

All strategies, including physicochemical techniques, require enhancermolecules that interact more or less directly with membranes andcomprise essentially small chemical molecules, secondary metabolites,peptides and proteins. A common feature of all these substances is thatthey are per se not target cell-specific and distribute with otherkinetics than the targeted toxins. This is one major drawback of thecurrent approaches.

The present invention will be described with respect to particularembodiments but the invention is not limited thereto but only by theclaims. The embodiments of the invention described herein can operate incombination and cooperation, unless specified otherwise.

While the invention has been described in terms of several embodiments,it is contemplated that alternatives, modifications, permutations andequivalents thereof will become apparent to one having ordinary skill inthe art upon reading the specification and upon study of the drawingsand graphs. The invention is not limited in any way to the illustratedembodiments. Changes can be made without departing from the scope whichis defined by the appended claims.

An aspect of the invention relates to a therapeutic molecule withchemical structure of COMPOUND I:

A1_(m)((-L9_(w))((-L1_(q)-B1_(n))_(u)((-L2_(r)-L3_(s))(-L4_(v)-C)_(p))_(t)))_(x)  (compound I),

whereinA1 is a first ligand if B1 is a first effector moiety, or A1 is thefirst effector moiety if B1 is the first ligand;C is a saponin;m=0 or 1 if A1 is the first ligand and B1 is the first effector moiety;m=0-32 if A1 is the first effector moiety and B1 is the first ligand;n=0 or 1 if B1 is the first ligand and A1 is the first effector moiety,or if A1 is the first ligand and B1 is the first effector moiety;p=any of 1-128;L1 is at least one linker for covalently coupling two chemical groups;L2 is at least one linker for covalently coupling two chemical groups;L3 is at least one oligomeric or polymeric scaffold for covalentlycoupling two chemical groups;L4 is at least one linker for covalently coupling two chemical groups;L9 is a tri-functional linker for covalently coupling three chemicalgroups;q=0 or 1;r=0 or 1;s=0 or 1;t=0, 1 or 2 if s=0, and t=any of 0-16 if s=1;u=any of 0-32 if A1 is the first ligand and B1 is the first effectormoiety, or u=1 if A1 is the first effector moiety and B1 is the firstligand;v=0 or 1;w=1 or 0; andx=1-16.

An aspect of the invention relates to a therapeutic combinationcomprising the therapeutic molecule according to the invention and asecond therapeutic molecule with chemical structure of COMPOUND II:

A2_(a)((-L10_(i))((-L5_(d)-B2_(b))_(h)((-L6_(e)-L7_(f))(-L8_(i)-C)_(c))_(q)))_(k)  (compound II),

whereinA2 is a second ligand if B2 is a second effector moiety, or A2 is thesecond effector moiety if B2 is the second ligand;C is a saponin;a=0 or 1 if A2 is the second ligand and B2 is the second effectormoiety, or a=0-32 if A2 is the second effector moiety and B2 is thesecond ligand;b=0 or 1 if B2 is the second ligand and A2 is the second effectormoiety, or if A2 is the second ligand and B2 is the second effectormoiety;c=any of 1-128;L5 is at least one linker for covalently coupling two chemical groups;L6 is at least one linker for covalently coupling two chemical groups;L7 is at least one oligomeric or polymeric scaffold for covalentlycoupling two chemical groups;L8 is at least one linker for covalently coupling two chemical groups;L10 is a tri-functional linker for covalently coupling three chemicalgroups;d=0 or 1;e=0 or 1;f=0 or 1;g=0, 1 or 2 if f=0, and g=any of 0-16 if f=1;h=any of 0-32 if A2 is the second ligand and B2 is the second effectormoiety, or h=1 if A2 is the second effector moiety and B2 is the secondligand;i=0 or 1;j=1 or 0 and;k=1-16.

An embodiment is the second therapeutic molecule of the invention,wherein g=0, 1 or 2 if f=0 and t>0, g=1 or 2 if f=0 and t=0, g=any of0-16 if f=1 and t>0, and g=any of 1-16 if f=1 and t=0.

An embodiment is the therapeutic molecule of the invention or the secondtherapeutic molecule of the invention, wherein the first ligand A1 or B1and/or the second ligand A2 or B2 comprise(s) or consist(s) of animmunoglobulin, a binding domain of an immunoglobulin or a bindingfragment of an immunoglobulin, such as an antibody, an IgG, a moleculecomprising or consisting of a Vhh domain or Vh domain, a Fab, an scFv,an Fv, a dAb, an F(ab)₂, Fcab fragment, or comprise(s) or consist(s) ofat least one non-proteinaceous ligand and/or at least one proteinaceousligand, the ligand for binding to a cell-surface molecule such as EGF ora cytokine, with the proviso that the first ligand and the second ligandare the same or are different.

An embodiment is the therapeutic molecule of the invention or the secondtherapeutic molecule of the invention, wherein the first ligand A1 or B1and/or the second ligand A2 or B2 bind(s) to a tumor-cell epitope,preferably a tumor-cell specific epitope, of a tumor-cell receptor,preferably a tumor-cell specific receptor, preferably selected fromCD71, CA125, EpCAM(17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin,syndecan-1, vascular integrin alpha-V beta-3, HER2, EGFR, CD20, CD22,Folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, PSMA,CanAg, integrin-alphaV, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239,CD70, CD123, CD352, DLL3, CD25, ephrinA4, MUC1, Trop2, CEACAM5, CEACAM6,HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1,CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2, more preferably selected from CD71,EGFR and HER2, with the proviso that the first ligand and the secondligand bind to the same or to a different tumor-cell epitope, preferablya tumor-cell specific epitope, and/or wherein the tumor-cell receptor,preferably the tumor-cell specific receptor, to which the first ligandcan bind is the same as, or is different from the tumor-cell receptor,preferably the tumor-cell specific receptor, to which the second ligandcan bind.

An embodiment is the therapeutic molecule of the invention or the secondtherapeutic molecule of the invention, wherein the first ligand A1 or B1and/or the second ligand A2 or B2 comprise(s) or consist(s) ofcetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab,brentuximab, inotuzumab, moxetumomab, polatuzumab, obinutuzumab, OKT-9anti-CD71 monoclonal antibody of the IgG type, pertuzumab, rituximab,ofatumumab, Herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38monoclonal antibody, an antibody of Table A2 or Table A3 or Table A4,preferably cetuximab or trastuzumab or OKT-9, or at least one tumor-cellreceptor binding-domain thereof and/or at least one tumor-cell receptorbinding-fragment thereof which are preferably (a) tumor-cell specificreceptor binding-domain(s) and/or (a) tumor-cell specific receptorbinding-fragment(s), with the proviso that the first ligand is the sameor different from the second ligand.

An embodiment is the therapeutic molecule of the invention or the secondtherapeutic molecule of the invention, wherein the first ligand A1 or B1is internalized by a tumor cell after binding of the first ligand to itsbinding partner on the tumor cell, and wherein preferably binding of thefirst ligand to the tumor cell is followed by tumor-cellreceptor-mediated internalization, e.g. via endocytosis, of a complex ofthe first ligand and the binding partner of the first ligand on thetumor cell.

An embodiment is the therapeutic molecule of the invention or the secondtherapeutic molecule of the invention, wherein the second ligand A2 orB2 is internalized by a tumor cell after binding of the second ligand toits binding partner on the tumor cell, and wherein preferably binding ofthe second ligand to the tumor cell is followed by tumor-cellreceptor-mediated internalization, e.g. via endocytosis, of a complex ofthe second ligand and the binding partner of the second ligand on thetumor cell.

An embodiment is the therapeutic molecule of the invention or the secondtherapeutic molecule of the invention, wherein the first effector moietyA1 or B1 and/or the second effector moiety A2 or B2 comprise(s) orconsist(s) of at least one of any one or more of an oligonucleotide, anucleic acid and a xeno nucleic acid, preferably selected from any oneor more of a vector, a gene, a cell suicide inducing transgene,deoxyribonucleic acid (DNA), ribonucleic acid (RNA), anti-senseoligonucleotide (ASO, AON), short interfering RNA (siRNA), microRNA(miRNA), DNA aptamer, RNA aptamer, mRNA, mini-circle DNA, peptidenucleic acid (PNA), phosphoramidate morpholino oligomer (PMO), lockednucleic acid (LNA), bridged nucleic acid (BNA),2′-deoxy-2′-fluoroarabino nucleic acid (FANA), 2′-O-methoxyethyl-RNA(MOE), 2′-0,4′-aminoethylene bridged nucleic acid, 3′-fluoro hexitolnucleic acid (FHNA), a plasmid, glycol nucleic acid (GNA) and threosenucleic acid (TNA), or a derivative thereof, more preferably a BNA, forexample a BNA for silencing HSP27 protein expression, with the provisothat the first effector moiety and the second effector moiety are thesame or are different.

An embodiment is the therapeutic molecule of the invention or the secondtherapeutic molecule of the invention, wherein the first effector moietyA1 or B1 and/or the second effector moiety A2 or B2 comprise(s) orconsist(s) of at least one proteinaceous molecule, preferably selectedfrom any one or more of a peptide, a protein, an enzyme such as ureaseand Cre-recombinase, a proteinaceous toxin, a ribosome-inactivatingprotein, at least one protein toxin selected from Table A5 and/or abacterial toxin, a plant toxin, more preferably selected from any one ormore of a viral toxin such as apoptin; a bacterial toxin such as Shigatoxin, Shiga-like toxin, Pseudomonas aeruginosa exotoxin (PE) orexotoxin A of PE, full-length or truncated diphtheria toxin (DT),cholera toxin; a fungal toxin such as alpha-sarcin; a plant toxinincluding ribosome-inactivating proteins and the A chain of type 2ribosome-inactivating proteins such as dianthin e.g. dianthin-30 ordianthin-32, saporin e.g. saporin-S3 or saporin-S6, bouganin orde-immunized derivative debouganin of bouganin, shiga-like toxin A,pokeweed antiviral protein, ricin, ricin A chain, modeccin, modeccin Achain, abrin, abrin A chain, volkensin, volkensin A chain, viscumin,viscumin A chain; or an animal or human toxin such as frog RNase, orgranzyme B or angiogenin from humans, or any fragment or derivativethereof; preferably the protein toxin is dianthin and/or saporin, withthe proviso that the first effector moiety/moieties and the secondeffector moiety/moieties are the same or are different.

An embodiment is the therapeutic molecule of the invention or the secondtherapeutic molecule of the invention, wherein the first effector moietyA1 or B1 and/or the second effector moiety A2 or B2 comprise(s) orconsist(s) of at least one payload, preferably selected from any one ormore of a toxin targeting ribosomes, a toxin targeting elongationfactors, a toxin targeting tubulin, a toxin targeting DNA and a toxintargeting RNA, more preferably any one or more of emtansine, pasudotox,maytansinoid derivative DM1, maytansinoid derivative DM4, monomethylauristatin E (MMAE, vedotin), monomethyl auristatin F (MMAF, mafodotin),a Calicheamicin, N-Acetyl-γ-calicheamicin, a pyrrolobenzodiazepine (PBD)dimer, a benzodiazepine, a CC-1065 analogue, a duocarmycin, Doxorubicin,paclitaxel, docetaxel, cisplatin, cyclophosphamide, etoposide,docetaxel, 5-fluorouracyl (5-FU), mitoxantrone, a tubulysin, anindolinobenzodiazepine, AZ13599185, a cryptophycin, rhizoxin,methotrexate, an anthracycline, a camptothecin analogue, SN-38,DX-8951f, exatecan mesylate, truncated form of Pseudomonas aeruginosaexotoxin (PE38), a Duocarmycin derivative, an amanitin, a-amanitin, aspliceostatin, a thailanstatin, ozogamicin, tesirine, Amberstatin269 andsoravtansine, or a derivative thereof.

An embodiment is the therapeutic molecule of the invention or the secondtherapeutic molecule of the invention, wherein the therapeutic moleculeand/or the second therapeutic molecule comprise(s) or consist(s) of anyone of Gemtuzumab ozogamicin, Brentuximab vedotin, Trastuzumabemtansine, Inotuzumab ozogamicin, Moxetumomab pasudotox and Polatuzumabvedotin and an antibody-drug conjugate of Table A2 and Table A3, or atleast one tumor-cell specific receptor binding-domain thereof and/or atleast one tumor-cell specific receptor binding-fragment thereof whichare preferably (a) tumor-cell specific receptor binding-domain(s) and/or(a) tumor-cell specific receptor binding-fragment(s), with the provisothat the therapeutic molecule and the second therapeutic molecule arethe same or are different.

An embodiment is the therapeutic molecule of the invention or the secondtherapeutic molecule of the invention, wherein the saponin C is atriterpenoid saponin or a bisdesmosidic triterpene saponin, belonging tothe type of a 12,13-dehydrooleanane with an aldehyde function inposition C-23 and optionally comprising a glucuronic acid function in acarbohydrate substituent at the C-3beta-OH group of the saponin, and/ora saponin isolated from a Gypsophila species and/or a Saponaria speciesand/or an Agrostemma species and/or a Quillaja species such as Quillajasaponaria.

An embodiment is the therapeutic molecule of the invention or the secondtherapeutic molecule of the invention, wherein the saponin C is a singlespecific saponin or is a mixture of two or more different saponins, suchas one or more of the saponins in Table A1 or Scheme I, SO1861, SA1657,GE1741, SA1641, QS-21, QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B, QS-21B-api, QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861,QS1862, Quillajasaponin, Saponinum album, QS-18, Quil-A, Gyp1, gypsosideA, AG1, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, or any of theirstereomers and/or any combinations thereof, preferably the saponin isSO1861 and/or GE1741 and/or SA1641 and/or QS-21 and/or saponin with aquillaic acid aglycon core, a Gal-(1→2)-[Xyl-(1→3)]-GlcA carbohydratesubstituent at the C-3beta-OH group and aGlc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuccarbohydrate substituent at the C-28-OH group, and/or is3-O-beta-D-galactopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)]-beta-D-glucuronopyranosylquillaic acid 28-O-beta-D-glucopyranosyl-(1→3)-beta-D-xylopyranosyl-(1→4)-alpha-L-rhamnopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)-4-OAc-beta-D-quinovopyranosyl-(1→4)]-beta-D-fucopyranoside,more preferably the saponin is SO1861 and/or QS-21.

An embodiment is the therapeutic molecule of the invention or the secondtherapeutic molecule of the invention, wherein the saponin C is abisdesmosidic saponin having a molecular mass of at least 1.500 Daltonand comprising an oleanan-type triterpene containing an aldehyde groupat the C-23 position and optionally a hydroxyl group at the C-16position, with a first branched carbohydrate side chain at the C-3position which first branched carbohydrate side chain optionallycontains glucuronic acid, wherein the saponin contains an ester groupwith a second branched carbohydrate side chain at the C-28 positionwhich second branched carbohydrate chain preferably comprises at leastfour carbohydrate units, optionally containing at least one acetylresidue such as two acetyl residues and/or at least one deoxycarbohydrates and/or a quinovose and/or a glucose and/or4-methoxycinnamic acid and/or optionally comprising5-O-[5-O-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoicacid and/or optionally comprising5-O-[5-O-Rha-(1→2)-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoicacid bound to a carbohydrate via an ester bond, or wherein the at leastone saponin is QS-21 or any one or more of QS-21A, QS-21 A-api, QS-21A-xyl, QS-21B, QS-21 B-api, QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api,QS-17-xyl, QS-18, QS1861, protonated QS1861 (QS1862), Quil-A.

An embodiment is the therapeutic molecule of the invention or the secondtherapeutic molecule of the invention, wherein the saponin C is abisdesmosidic triterpene saponin belonging to the type of a12,13-dehydrooleanane with an aldehyde function in position C-23,wherein the saponin C is covalently coupled to an amino-acid residue ofthe first ligand A1 or B1 and/or the first effector moiety B1 or A1and/or the second ligand A2 or B2 and/or the second effector moiety B2or A2 via the aldehyde function in the saponin C, preferably saidaldehyde function in position C-23, preferably via a linker L2, L4, L6,L8, L9 and/or L10, more preferably via a cleavable linker L2, L4, L6,L8, L9 and/or L10, wherein the amino-acid residue preferably is selectedfrom cysteine and lysine.

An embodiment is the therapeutic molecule of the invention or the secondtherapeutic molecule of the invention, wherein the saponin C is abisdesmosidic triterpene saponin belonging to the type of a12,13-dehydrooleanane with an aldehyde function in position C-23,wherein the aldehyde function in position C-23 of the at least onesaponin is covalently coupled to linker N-ε-maleimidocaproic acidhydrazide, which linker is covalently coupled via a thio-ether bond to asulfhydryl group in the first ligand A1 or B1 and/or in the firsteffector moiety B1 or A1 and/or in the second ligand A2 or B2 and/or inthe second effector moiety B2 or A2, such as a sulfhydryl group of acysteine.

An embodiment is the therapeutic molecule of the invention or the secondtherapeutic molecule of the invention, wherein the saponin C is abisdesmosidic triterpene saponin belonging to the type of a12,13-dehydrooleanane with an aldehyde function in position C-23 andcomprising a glucuronic acid function in a carbohydrate substituent atthe C-3beta-OH group of the saponin, wherein the saponin C is covalentlycoupled to the amino-acid residue of the first ligand A1 or B1 and/orthe first effector moiety B1 or A1 and/or the second ligand A2 or B2and/or the second effector moiety B2 or A2 via the glucuronic acidfunction in the saponin C, if present, preferably via a linker L2, L4,L6, L8, L9 and/or L10, wherein the amino-acid residue preferably isselected from cysteine and lysine, more preferably the amino-acidresidue is lysine.

An embodiment is the therapeutic molecule of the invention or the secondtherapeutic molecule of the invention, wherein the saponin C is abisdesmosidic triterpene saponin belonging to the type of a12,13-dehydrooleanane with an aldehyde function in position C-23 andcomprising a glucuronic acid function in a carbohydrate substituent atthe C-3beta-OH group of the saponin, wherein the glucuronic acidfunction in the carbohydrate substituent at the C-3beta-OH group of theat least one saponin is covalently coupled to linker1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate, which linker is covalently coupled via anamide bond to an amine group in the first ligand A1 or B1 and/or in thefirst effector moiety B1 or A1 and/or in the second ligand A2 or B2and/or in the second effector moiety B2 or A2, such as an amine group ofa lysine or an N-terminus of the first ligand A1 or B1 and/or the firsteffector moiety B1 or A1 and/or the second ligand A2 or B2 and/or thesecond effector moiety B2 or A2.

An embodiment is the therapeutic molecule of the invention or the secondtherapeutic molecule of the invention, wherein the first ligand A1 or B1and/or the first effector moiety B1 or A1 and/or the second ligand A2 orB2 and/or the second effector moiety B2 or A2 comprise(s) one or morethan one covalently bound saponin C, preferably 2, 3, 4, 5, 6, 8, 10,16, 32, 64, 128 or 1-100 saponins, or any number of saponins thereinbetween, such as 7, 9, 12 saponins.

An embodiment is the therapeutic molecule of the invention or the secondtherapeutic molecule of the invention, wherein the first ligand A1 or B1and/or the first effector moiety B1 or A1 and/or the second ligand A2 orB2 and/or the second effector moiety B2 or A2 comprise(s) one or morethan one covalently bound saponin C, wherein the saponin(s) C is/arecovalently bound directly to an amino-acid residue of the first ligandA1 or B1 and/or the first effector moiety B1 or A1 and/or the secondligand A2 or B2 and/or the second effector moiety B2 or A2 when r, s, v,e, f and i are 0, preferably to a cysteine and/or to a lysine, and/oris/are covalently bound via at least one linker L2, L4, L6, L8, L9and/or L10, or via at least one cleavable linker L2, L4, L6, L8, L9and/or L10 and/or via at least one oligomeric or polymeric scaffold L3and/or L7, preferably 1-8 of such scaffolds or 2-4 of such scaffolds,wherein the at least one scaffold is optionally based on a dendron,wherein 1-32 saponins, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32 saponins,or any number of saponins therein between, such as 7, 9, 12 saponins,are covalently bound to the at least one scaffold.

An embodiment is the therapeutic molecule of the invention or the secondtherapeutic molecule of the invention, wherein the saponin C is abisdesmosidic triterpene saponin belonging to the type of a12,13-dehydrooleanane with an aldehyde function in position C-23,wherein the saponin C is covalently coupled to an amino-acid residue ofthe first ligand A1 or B1 and/or the first effector moiety B1 or A1and/or the second ligand A2 or B2 and/or the second effector moiety B2or A2 via the aldehyde function in the saponin C, preferably saidaldehyde function in position C-23, preferably via a linker L2, L4, L6,L8, L9 and/or L10, more preferably via a cleavable linker L2, L4, L6,L8, L9 and/or L10, wherein the amino-acid residue preferably is selectedfrom cysteine and lysine, and wherein the cleavable linker L2, L4, L6,L8, L9 and/or L10 is subject to cleavage under acidic conditions,reductive conditions, enzymatic conditions or light-induced conditions,and preferably the cleavable linker comprises a hydrazone bond or ahydrazide bond subject to cleavage under acidic conditions when bound tosaponin, and/or comprises a bond susceptible to proteolysis, for exampleproteolysis by Cathepsin B, when bound to saponin, and/or the cleavablelinker comprises a disulphide bond susceptible to cleavage underreductive conditions.

An embodiment is the therapeutic molecule of the invention or the secondtherapeutic molecule of the invention, wherein the saponin C is abisdesmosidic triterpene saponin belonging to the type of a12,13-dehydrooleanane with an aldehyde function in position C-23,wherein the saponin C is covalently coupled to an amino-acid residue ofthe first ligand A1 or B1 and/or the first effector moiety B1 or A1and/or the second ligand A2 or B2 and/or the second effector moiety B2or A2 via the aldehyde function in the saponin C, preferably saidaldehyde function in position C-23, preferably via a linker L2, L4, L6,L8, L9 and/or L10, more preferably via a cleavable linker L2, L4, L6,L8, L9 and/or L10, wherein the amino-acid residue preferably is selectedfrom cysteine and lysine, and wherein the cleavable linker L2, L4, L6,L8, L9 and/or L10 is subject to cleavage in vivo under acidic conditionsas present in endosomes and/or lysosomes of mammalian cells, preferablyhuman cells, preferably at pH 4.0-6.5, and more preferably at pH≤5.5.

An embodiment is the therapeutic molecule of the invention or the secondtherapeutic molecule of the invention, wherein the polymeric oroligomeric scaffold L3 and/or L7 comprises a polymeric or oligomericstructure and comprises a chemical group, the chemical group forcovalently coupling of the polymeric or oligomeric scaffold L3 and/or L7to the amino-acid residue of the first ligand and/or the first effectormoiety and/or the second ligand and/or the second effector moiety.

An embodiment is the therapeutic molecule of the invention or the secondtherapeutic molecule of the invention, wherein the saponin C is abisdesmosidic triterpene saponin belonging to the type of a12,13-dehydrooleanane with an aldehyde function in position C-23 andcomprising a glucuronic acid function in a carbohydrate substituent atthe C-3beta-OH group of the saponin, wherein the glucuronic acidfunction in the carbohydrate substituent at the C-3beta-OH group of theat least one saponin is covalently coupled to linker1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate, which linker is covalently coupled via anamide bond to an amine group in the first ligand A1 or B1 and/or in thefirst effector moiety B1 or A1 and/or in the second ligand A2 or B2and/or in the second effector moiety B2 or A2, such as an amine group ofa lysine or an N-terminus of the first ligand A1 or B1 and/or the firsteffector moiety B1 or A1 and/or the second ligand A2 or B2 and/or thesecond effector moiety B2 or A2, and wherein the at least one saponin iscovalently bound to the polymeric or oligomeric structure of thescaffold L3 and/or L7 via a cleavable linker L4 and/or L8 according tothe invention.

An embodiment is the therapeutic molecule of the invention or the secondtherapeutic molecule of the invention, wherein the chemical group of thepolymeric or oligomeric scaffold L3 and/or L7, for covalently couplingof the scaffold to the amino-acid residue of the first ligand and/or thefirst effector moiety and/or the second ligand and/or the secondeffector moiety, is a click chemistry group, preferably selected from atetrazine, an azide, an alkene or an alkyne, or a cyclic derivative ofthese groups, more preferably the click chemistry group is an azide.

An embodiment is the therapeutic molecule of the invention or the secondtherapeutic molecule of the invention, wherein the first ligand A1 or B1and/or the first effector moiety B1 or A1 and/or the second ligand A2 orB2 and/or the second effector moiety B2 or A2 comprise(s) one or morethan one covalently bound saponin C, preferably 2, 3, 4, 5, 6, 8, 10,16, 32, 64, 128 or 1-100 saponins, or any number of saponins thereinbetween, such as 7, 9, 12 saponins, and wherein the at least one saponinis covalently bound to the first ligand and/or to the first effectormoiety and/or to the second ligand and/or to the second effector moiety,either directly or via at least one linker such as a bi-functionallinker, for example based on N-ε-maleimidocaproic acid hydrazide and/orbased on1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate, or a tri-functional linker L9 when w=1and/or a tri-functional linker L10 when j=1, such as the tri-functionallinker of Scheme II.

An embodiment is the therapeutic molecule of the invention andencompassing the previous embodiment, or the second therapeutic moleculeof the invention encompassing the previous embodiment, wherein thetri-functional linker L9 when w=1 and/or the tri-functional linker L10when j=1, comprises a second chemical group with at least one saponincovalently bound thereto, a third chemical group for covalent binding tothe first and/or second ligand and a first chemical group for covalentbinding to at least one first and/or second effector moiety, preferablythe tri-functional linker is the trifunctional linker of Scheme II.

An embodiment is the therapeutic molecule of the invention or the secondtherapeutic molecule of the invention, wherein the at least one saponinis covalently bound to the first ligand and/or to the first effectormoiety and/or to the second ligand and/or to the second effector moietyvia at least one linker comprising a tri-functional linker L9 when j=1and/or a tri-functional linker L10 when w=1, to which tri-functionallinker both the first ligand and the at least one first effector moietyare bound and/or to which tri-functional linker both the second ligandand the at least one second effector moiety are bound, preferably thetri-functional linker is the trifunctional linker of Scheme II.

An embodiment is the therapeutic molecule of the invention or the secondtherapeutic molecule of the invention, wherein the polymeric oroligomeric structure of the scaffold L3 and/or L7 comprises a linear,branched and/or cyclic polymer, oligomer, dendrimer, dendron,dendronized polymer, dendronized oligomer, a DNA, a polypeptide,poly-lysine, a poly-ethylene glycol, or an assembly of these polymericor oligomeric structures which assembly is preferably built up bycovalent cross-linking.

An embodiment is the therapeutic molecule of the invention or the secondtherapeutic molecule of the invention, wherein the first ligand A1 or B1and/or the first effector moiety B1 or A1 and/or the second ligand A2 orB2 and/or the second effector moiety B2 or A2 comprise(s) one or morethan one covalently bound saponin C, preferably 2, 3, 4, 5, 6, 8, 10,16, 32, 64, 128 or 1-100 saponins, or any number of saponins thereinbetween, such as 7, 9, 12 saponins, and wherein the first ligand A1 orB1 is covalently bound to the first effector moiety B1 or A1,respectively, via at least one linker L1, and/or wherein the secondligand A2 or B2 is covalently bound to the second effector moiety B2 orA2, respectively, via at least one linker L5.

An embodiment is the therapeutic combination of the invention, whereinthe first ligand A1 is a monoclonal antibody or at least one bindingfragment or -domain thereof according to any one of the claims 3-7, m=1,q=0, n=0, u=0, r=0, L3 is the scaffold according to the invention ands=1, or L3 is absent and s=0, L4 is a linker or a cleavable linkeraccording to the invention, v=1, p=2-4 and t=2-4 if s=1 and t=0 if s=0,and saponin C is a saponin according to the invention, preferably thesaponin C is SO1861 and/or QS-21, and effector moiety A2 is an effectormoiety selected from any one or more of an oligonucleotide, a nucleicacid and a xeno nucleic acid, preferably selected from any one or moreof a vector, a gene, a cell suicide inducing transgene, deoxyribonucleicacid (DNA), ribonucleic acid (RNA), anti-sense oligonucleotide (ASO,AON), short interfering RNA (siRNA), microRNA (miRNA), DNA aptamer, RNAaptamer, mRNA, mini-circle DNA, peptide nucleic acid (PNA),phosphoramidate morpholino oligomer (PMO), locked nucleic acid (LNA),bridged nucleic acid (BNA), 2′-deoxy-2′-fluoroarabino nucleic acid(FANA), 2′-O-methoxyethyl-RNA (MOE), 2′-0,4′-aminoethylene bridgednucleic acid, 3′-fluoro hexitol nucleic acid (FHNA), a plasmid, glycolnucleic acid (GNA) and threose nucleic acid (TNA), or a derivativethereof, more preferably a BNA, for example a BNA for silencing HSP27protein expression, a=1, d=0, b=0, h=0, e=0, f=0, i=0, c=0 and g=0.

An embodiment is the therapeutic molecule of the invention, wherein r=0,s=0, v=0, s=0, v=0, p=0, t=0, ligand A1 is a monoclonal antibody or atleast one binding fragment or -domain thereof according to any one ofthe invention, m=1, effector moiety B1 is an effector moiety selectedfrom any one or more of an oligonucleotide, a nucleic acid and a xenonucleic acid, preferably selected from any one or more of a vector, agene, a cell suicide inducing transgene, deoxyribonucleic acid (DNA),ribonucleic acid (RNA), anti-sense oligonucleotide (ASO, AON), shortinterfering RNA (siRNA), microRNA (miRNA), DNA aptamer, RNA aptamer,mRNA, mini-circle DNA, peptide nucleic acid (PNA), phosphoramidatemorpholino oligomer (PMO), locked nucleic acid (LNA), bridged nucleicacid (BNA), 2′-deoxy-2′-fluoroarabino nucleic acid (FANA),2′-O-methoxyethyl-RNA (MOE), 2′-0,4′-aminoethylene bridged nucleic acid,3′-fluoro hexitol nucleic acid (FHNA), a plasmid, glycol nucleic acid(GNA) and threose nucleic acid (TNA), or a derivative thereof, morepreferably a BNA, for example a BNA for silencing HSP27 proteinexpression, either q=0, n=1 and u=2-4, or q=1, n=2-4, u=2-4 and linkerL1 is the oligomeric or polymeric scaffold L3 according to theinvention.

An embodiment is the therapeutic combination of the invention, whereinthe therapeutic molecule is the therapeutic molecule of the previousembodiment, and wherein the second ligand A2 is a monoclonal antibody orat least one binding fragment or -domain thereof according to theinvention, a=1, d=0, b=0, h=0, e=0, L7 is the scaffold according to theinvention and f=1, or L7 is absent and f=0, L8 is a linker or acleavable linker according to the invention, i=1, c=2-4 and g=2-4 if f=1and g=0 if f=0, and saponin C is a saponin according to the invention,preferably the saponin C is SO1861 and/or QS-21, with the proviso thatthe ligand A1 and the ligand A2 are the same or are different.

An aspect of the invention relates to a therapeutic combination, whereinthe therapeutic combination comprises: (a) a first pharmaceuticalcomposition comprising the therapeutic molecule with chemical structureof COMPOUND I according to the invention, the first pharmaceuticalcomposition optionally further comprising a pharmaceutically acceptableexcipient; and (b) a second pharmaceutical composition comprising thesecond therapeutic molecule with chemical structure of COMPOUND IIaccording to the invention, the second pharmaceutical compositionoptionally further comprising a pharmaceutically acceptable excipient.

An aspect of the invention relates to the first pharmaceuticalcomposition of the invention for use as a medicament.

An aspect of the invention relates to a therapeutic combination for usein the treatment or prevention of cancer in a human subject, wherein thetherapeutic combination comprises: (a) the first pharmaceuticalcomposition of the invention; and (b) the second pharmaceuticalcomposition of the invention, wherein the ligand A1 or B1 and the ligandA2 or B2 can bind to a tumor-cell epitope, preferably to a tumor-cellspecific epitope, on a tumor-cell surface molecule, preferably on atumor cell-specific surface molecule, with the proviso that thetumor-cell epitope or tumor-cell specific epitope to which the ligand A1or B1 can bind is the same as, or is different from the tumor-cellepitope or the tumor-cell specific epitope to which the ligand A2 or B2can bind.

An aspect of the invention relates to the first pharmaceuticalcomposition of the invention, for use in the treatment or prophylaxis ofcancer in a patient in need thereof, wherein the ligand A1 or B1 canbind to a tumor-cell epitope, preferably a tumor-cell specific epitope,on a tumor-cell surface molecule, preferably a tumor cell-specificsurface molecule.

An embodiment is the first pharmaceutical composition for use accordingto the invention or the therapeutic combination for use according to theinvention, wherein the second pharmaceutical composition of theinvention and the first pharmaceutical composition of the invention areadministered to the patient in need thereof.

An aspect of the invention relates to the first pharmaceuticalcomposition of the invention further comprising the second therapeuticmolecule of the invention.

An aspect of the invention relates to the first pharmaceuticalcomposition of the invention further comprising the second therapeuticmolecule of the invention, for use as a medicament.

An aspect of the invention relates to the first pharmaceuticalcomposition of the invention further comprising the second therapeuticmolecule of the invention, for use in the treatment or prevention of acancer in a human subject.

An aspect of the invention within a first series of aspects andembodiments of the invention relates to a scaffold suitable forcovalently binding at least one biologically active molecule to acarrier molecule, the scaffold comprising a polymeric or oligomericstructure and at least one of said biologically active moleculescovalently bound to said polymeric or oligomeric structure, wherein thescaffold further comprises a first chemical group for covalentlycoupling of the scaffold to the carrier molecule.

An embodiment within the first series of aspects and embodiments of theinvention is the scaffold according to the invention, wherein the atleast one biologically active molecule has a molecular mass of 3.000Dalton or less, preferably 2.500 Dalton or less, more preferably 2.300Dalton or less, most preferably, 2.000 Dalton or less, such as 1.700Dalton-1.950 Dalton.

An embodiment within the first series of aspects and embodiments of theinvention is the scaffold according to the invention, wherein the atleast one biologically active molecule is an amphiphilic molecule.

An embodiment within the first series of aspects and embodiments of theinvention is the scaffold according to the invention, wherein the atleast one biologically active molecule is a single specific molecule oris a mixture of different molecules, when more than one biologicallyactive molecules are covalently bound to the polymeric or oligomericstructure comprised by the scaffold.

An embodiment within the first series of aspects and embodiments of theinvention is the scaffold according to the invention, wherein the atleast one biologically active molecule is a glycoside, preferably abisdesmosidic triterpene or triterpenoid saponin, more preferably abisdesmosidic triterpene saponin, most preferably a bisdesmosidictriterpene saponin belonging to the type of a 12,13-dehydrooleanane withan aldehyde function in position C-23 and optionally comprising aglucuronic acid function in a carbohydrate substituent at the C-3beta-OHgroup of the saponin.

An embodiment within the first series of aspects and embodiments of theinvention is the scaffold according to the invention, wherein the atleast one biologically active molecule is a saponin that can be isolatedfrom a Gypsophila species and/or a Saponaria species and/or anAgrostemma species and/or a Quillaja species such as Quillaja saponariaor is a single specific saponin or is a mixture of two or more differentsaponins, such as one or more of the saponins in Table A1 or Scheme I,SO1861, SA1657, GE1741, SA1641, QS-21, QS-21A, QS-21 A-api, QS-21 A-xyl,QS-21B, QS-21 B-api, QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api,QS-17-xyl, QS1861, QS1862, Quillajasaponin, Saponinum album, QS-18,Quil-A, Gyp1, gypsoside A, AG1, AG2, SO1542, SO1584, SO1658, SO1674,SO1832, or any of their stereomers and/or any combinations thereof,preferably the saponin is SO1861 and/or GE1741 and/or SA1641 and/orQS-21 and/or saponin with a quillaic acid aglycon core, aGal-(1→2)-[Xyl-(1→3)]-GlcA carbohydrate substituent at the C-3beta-OHgroup and aGlc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuccarbohydrate substituent at the C-28-OH group, and/or is3-O-beta-D-galactopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)]-beta-D-glucuronopyranosylquillaic acid28-O-beta-D-glucopyranosyl-(1→3)-beta-D-xylopyranosyl-(1→4)-alpha-L-rhamnopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)-4-OAc-beta-D-quinovopyranosyl-(1→4)]-beta-D-fucopyranoside,more preferably the saponin is SO1861 and/or QS-21.

An embodiment within the first series of aspects and embodiments of theinvention is the scaffold according to the invention, wherein the atleast one biologically active molecule is a bisdesmosidic saponin havinga molecular mass of at least 1.500 Dalton and comprising an oleanan-typetriterpene containing an aldehyde group at the C-23 position andoptionally a hydroxyl group at the C-16 position, with a first branchedcarbohydrate side chain at the C-3 position which first branchedcarbohydrate side chain optionally contains glucuronic acid, wherein thesaponin contains an ester group with a second branched carbohydrate sidechain at the C-28 position which second branched carbohydrate chainpreferably comprises at least four carbohydrate units, optionallycontaining at least one acetyl residue such as two acetyl residuesand/or optionally comprising deoxy carbohydrates and/or optionallycomprising quinovose and/or optionally comprising glucose and/oroptionally comprising 4-methoxycinnamic acid and/or optionallycomprising5-O-[5-O-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoicacid and/or optionally comprising5-O-[5-O-Rha-(1→2)-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoicacid bound to a carbohydrate via an ester bond, or wherein the at leastone saponin is QS-21 or any one or more of QS-21A, QS-21 A-api, QS-21A-xyl, QS-21B, QS-21 B-api, QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api,QS-17-xyl, QS-18, QS1861, protonated QS1861 (QS1862), Quil-A.

An embodiment within the first series of aspects and embodiments of theinvention is the scaffold according to the invention, wherein the atleast one biologically active molecule is covalently bound to thepolymeric or oligomeric structure via a non-cleavable bond or via acleavable bond, wherein preferably said cleavable bond is subject tocleavage under acidic conditions, reductive conditions, enzymaticconditions or light-induced conditions, more preferably the cleavablebond is a hydrazone bond or a hydrazide bond subject to cleavage underacidic conditions, and/or is a bond susceptible to proteolysis, forexample proteolysis by Cathepsin B, and/or is a bond susceptible forcleavage under reductive conditions such as a disulphide bond.

An embodiment within the first series of aspects and embodiments of theinvention is the scaffold according to the invention, wherein the atleast one biologically active molecule is covalently bound to thepolymeric or oligomeric structure via a cleavable bond, wherein saidcleavable bond is subject to cleavage in vivo under acidic conditions aspresent in endosomes and/or lysosomes of mammalian cells, preferablyhuman cells, preferably at pH 4.0-6.5, and more preferably at pH≤5.5.

An embodiment within the first series of aspects and embodiments of theinvention is the scaffold according to the invention, wherein the atleast one biologically active molecule is covalently bound to thepolymeric or oligomeric structure of the scaffold via an imine bond, ahydrazone bond, a hydrazide bond, an oxime bond, a 1,3-dioxolane bond, adisulphide bond, a thio-ether bond, an amide bond, a peptide bond or anester bond, preferably via at least one linker.

An embodiment within the first series of aspects and embodiments of theinvention is the scaffold according to the invention, wherein thealdehyde function in position C-23 of the at least one saponin isinvolved in the covalent bonding to the polymeric or oligomericstructure of the scaffold, and/or, if present, the glucuronic acidfunction in the carbohydrate substituent at the C-3beta-OH group of theat least one saponin, is involved in the covalent bonding to thepolymeric or oligomeric structure of the scaffold, either via directbinding or via at least one linker.

An embodiment within the first series of aspects and embodiments of theinvention is the scaffold according to the invention, wherein thealdehyde function in position C-23 of the at least one saponin iscovalently coupled to linker N-ε-maleimidocaproic acid hydrazide, whichlinker is covalently coupled via a thio-ether bond to a sulfhydryl groupin the polymeric or oligomeric structure of the scaffold, such as asulfhydryl group of a cysteine.

An embodiment within the first series of aspects and embodiments of theinvention is the scaffold according to the invention, wherein theglucuronic acid function in the carbohydrate substituent at theC-3beta-OH group of the at least one saponin is covalently coupled tolinker1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate, which linker is covalently coupled via anamide bond to an amine group in the polymeric or oligomeric structure ofthe scaffold, such as an amine group of a lysine or an N-terminus of aproteinaceous molecule.

An embodiment within the first series of aspects and embodiments of theinvention is the scaffold according to the invention, wherein thechemical group for covalently coupling of the scaffold to the carriermolecule is a click chemistry group.

An embodiment within the first series of aspects and embodiments of theinvention is the scaffold according to the invention, wherein the clickchemistry group is a tetrazine, an azide, an alkene or an alkyne, or acyclic derivative of any of these groups, preferably an azide.

An embodiment within the first series of aspects and embodiments of theinvention is the scaffold according to the invention, wherein thescaffold is a tri-functional linker comprising a second chemical groupwith at least one biologically active molecule covalently bound thereto,comprising a third chemical group for covalent binding to a molecule andcomprising the first chemical group for covalent binding to the carrier,preferably the tri-functional linker is the tri-functional linker ofScheme II and Structure B.

An embodiment within the first series of aspects and embodiments of theinvention is the scaffold according to the invention, wherein the atleast one biologically active molecule is a defined number of glycosidemolecules or a defined range of glycoside molecules, preferably 1-128 orat least 2, 3, 4, 5, 6, 8, 10, 16, 32, 64 or 128 glycoside molecules, orany number of glycoside molecules therein between, such as 7, 9, 12glycoside molecules.

An embodiment within the first series of aspects and embodiments of theinvention is the scaffold according to the invention, wherein thepolymeric or oligomeric structure comprises a linear, branched and/orcyclic polymer, oligomer, dendrimer, dendron, dendronized polymer,dendronized oligomer, a DNA, a polypeptide, a poly-lysine, apoly-ethylene glycol, or an assembly of these polymeric or oligomericstructures which assembly is preferably built up by covalentcross-linking.

An embodiment within the first series of aspects and embodiments of theinvention is the scaffold according to the invention, wherein thecarrier molecule comprises or consists of any of a proteinaceousmolecule, a protein, a peptide, a nucleic acid, an oligonucleotide, alipid, a fat, a fatty acid, a nanoparticle, a carbohydrate, or anycovalently bound conjugate or covalently bound complex of combinationsthereof.

An embodiment within the first series of aspects and embodiments of theinvention is the scaffold according to the invention, wherein thecarrier molecule comprises or consists of an immunoglobulin, at leastone binding domain of an immunoglobulin and/or at least one bindingfragment of an immunoglobulin, such as an antibody, an IgG, a moleculecomprising or consisting of a Vhh domain or Vh domain, a Fab, an scFv,an Fv, a dAb, an F(ab)₂, Fcab fragment, or comprises or consists of atleast one non-proteinaceous ligand and/or at least one proteinaceousligand for binding to a cell-surface molecule such as EGF or a cytokine.

An embodiment within the first series of aspects and embodiments of theinvention is the scaffold according to the invention, wherein thecarrier molecule comprises or consists of at least one binding domainand/or at least one binding fragment for binding to a cell-surfacereceptor such as a tumor-cell specific cell-surface receptor selectedfrom CD71, CA125, EpCAM(17-1A), CD52, CEA, CD44v6, FAP, EGF-IR,integrin, syndecan-1, vascular integrin alpha-V beta-3, HER2, EGFR,CD20, CD22, Folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor,PSMA, CanAg, integrin-alphaV, CA6, CD33, mesothelin, Cripto, CD3, CD30,CD239, CD70, CD123, CD352, DLL3, CD25, ephrinA4, MUC1, Trop2, CEACAM5,CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7,PD-L1, CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2, preferably selected fromCD71, EGFR, HER2.

An embodiment within the first series of aspects and embodiments of theinvention is the scaffold according to the invention, wherein thecarrier molecule comprises or consists of any one of cetuximab,daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab,inotuzumab, moxetumomab, polatuzumab, obinutuzumab, OKT-9 anti-CD71monoclonal antibody of the IgG type, pertuzumab, rituximab, ofatumumab,Herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonalantibody, an antibody of Table A2 or Table A3 or Table A4, preferablycetuximab or trastuzumab or OKT-9, or at least one tumor-cell receptorbinding-fragment thereof and/or at least one tumor-cell receptorbinding-domain thereof, such as at least one tumor-cell specificreceptor binding-fragment thereof and/or at least one tumor-cellspecific receptor binding-domain thereof.

An embodiment within the first series of aspects and embodiments of theinvention is the scaffold according to the invention, wherein thescaffold is suitable for forming a covalent bond with the carriermolecule, said covalent bond preferably involving a cysteine side-chainof the carrier molecule and/or a lysine side-chain of the carriermolecule when the carrier molecule comprises at least a cysteine and/ora lysine.

An embodiment within the first series of aspects and embodiments of theinvention is the scaffold according to the invention, wherein thecarrier molecule comprises or consists of at least one effectormolecule, or wherein the carrier further comprises at least one effectormolecule, wherein the effector molecule is at least one of an activepharmaceutical substance, such as any one or more of a payload, a toxin,a drug, a polypeptide, an oligonucleotide, a nucleic acid, a xenonucleic acid, an enzyme such as urease and Cre-recombinase, a proteintoxin, a ribosome-inactivating protein.

An embodiment within the first series of aspects and embodiments of theinvention is the scaffold according to the invention, wherein theprotein toxin comprises or consists of any one or more of a proteintoxin selected from Table A5 and/or a viral toxin such as apoptin; abacterial toxin such as Shiga toxin, Shiga-like toxin, Pseudomonasaeruginosa exotoxin (PE) or exotoxin A of PE, full-length or truncateddiphtheria toxin (DT), cholera toxin; a fungal toxin such asalpha-sarcin; a plant toxin including ribosome-inactivating proteins andthe A chain of type 2 ribosome-inactivating proteins such as dianthine.g. dianthin-30 or dianthin-32, saporin e.g. saporin-S3 or saporin-S6,bouganin or de-immunized derivative debouganin of bouganin, shiga-liketoxin A, pokeweed antiviral protein, ricin, ricin A chain, modeccin,modeccin A chain, abrin, abrin A chain, volkensin, volkensin A chain,viscumin, viscumin A chain; or an animal or human toxin such as frogRNase, or granzyme B or angiogenin from humans, or any fragment orderivative thereof; preferably the protein toxin is dianthin and/orsaporin.

An embodiment within the first series of aspects and embodiments of theinvention is the scaffold according to the invention, wherein theoligonucleotide, the xeno nucleic acid or the nucleic acid comprises orconsists of any one or more of a vector, a gene, a cell suicide inducingtransgene, deoxyribonucleic acid (DNA), ribonucleic acid (RNA),anti-sense oligonucleotide (ASO, AON), short interfering RNA (siRNA),microRNA (miRNA), DNA aptamer, RNA aptamer, mRNA, mini-circle DNA,peptide nucleic acid (PNA), phosphoramidate morpholino oligomer (PMO),locked nucleic acid (LNA), bridged nucleic acid (BNA),2′-deoxy-2′-fluoroarabino nucleic acid (FANA), 2′-O-methoxyethyl-RNA(MOE), 2′-0,4′-aminoethylene bridged nucleic acid, 3′-fluoro hexitolnucleic acid (FHNA), a plasmid, glycol nucleic acid (GNA) and threosenucleic acid (TNA), or a derivative thereof, preferably a BNA, forexample a BNA for silencing HSP27 protein expression.

An embodiment within the first series of aspects and embodiments of theinvention is the scaffold according to the invention, wherein theeffector molecule comprises or consists of at least one payload,preferably selected from any one or more of a toxin targeting ribosomes,a toxin targeting elongation factors, a toxin targeting tubulin, a toxintargeting DNA and a toxin targeting RNA, more preferably any one or moreof emtansine, pasudotox, maytansinoid derivative DM1, maytansinoidderivative DM4, monomethyl auristatin E (MMAE, vedotin), monomethylauristatin F (MMAF, mafodotin), a Calicheamicin,N-Acetyl-γ-calicheamicin, a pyrrolobenzodiazepine (PBD) dimer, abenzodiazepine, a CC-1065 analogue, a duocarmycin, Doxorubicin,paclitaxel, cisplatin, cyclophosphamide, etoposide, docetaxel,5-fluorouracyl (5-FU), mitoxantrone, a tubulysin, anindolinobenzodiazepine, AZ13599185, a cryptophycin, rhizoxin,methotrexate, an anthracycline, a camptothecin analogue, SN-38,DX-8951f, exatecan mesylate, truncated form of Pseudomonas aeruginosaexotoxin (PE38), a Duocarmycin derivative, an amanitin, a-amanitin, aspliceostatin, a thailanstatin, ozogamicin, tesirine, Amberstatin269 andsoravtansine, or a derivative thereof.

An embodiment within the first series of aspects and embodiments of theinvention is the scaffold according to the invention, wherein thecarrier molecule comprises or consists of a covalently linkedcombination of an effector molecule and a monoclonal antibody,preferably selected from Gemtuzumab ozogamicin, Brentuximab vedotin,Trastuzumab emtansine, Inotuzumab ozogamicin, Moxetumomab pasudotox andPolatuzumab vedotin and an antibody-drug conjugate of Table A2 and TableA3.

An aspect of the invention within the first series of aspects andembodiments of the invention relates to a method for producing ascaffold suitable for covalently binding at least one biologicallyactive molecule to a carrier molecule, the method comprising: a)providing a polymeric or oligomeric structure comprising a firstchemical group for covalently coupling of the polymeric structure or theoligomeric structure to the carrier molecule and comprising at least oneof a second chemical group different from the first chemical group,wherein each second chemical group is for covalently coupling one of theat least one biologically active molecules to the oligomeric orpolymeric structure; and b) covalently coupling at least onebiologically active molecule to said polymeric or oligomeric structurevia the second chemical group(s), wherein preferably the biologicallyactive molecule(s) is/are any one of the biologically active moleculesof the invention, more preferably SO1861 and/or GE1741 and/or SA1641and/or QS-21, therewith providing the scaffold.

An embodiment within the first series of aspects and embodiments of theinvention is the method according to the invention, the scaffoldcomprising at least one covalently bound biologically active molecule,the method comprising: a) providing a scaffold comprising at least onebiologically active molecule covalently bound to a polymeric oroligomeric structure in said scaffold, preferably providing a scaffoldaccording to the invention or the scaffold obtainable by the methodaccording to the invention or the scaffold obtained with the methodaccording to the invention; and b) covalently coupling the scaffold ofa) to a carrier molecule according to the invention, therewith providingthe scaffold covalently bound to a carrier molecule, the scaffoldcomprising at least one covalently bound biologically active molecule.

An embodiment within the first series of aspects and embodiments of theinvention is the method according to the invention, wherein the scaffoldis able to augment endosomal escape and/or lysosomal escape of theeffector molecule according to the invention when either said effectormolecule is covalently bound to the scaffold and contacted with amammalian cell, or when said effector molecule is contacted with amammalian cell in the presence of the scaffold.

An aspect of the invention within a second series of aspects andembodiments of the invention relates to a first proteinaceous moleculecomprising a first binding site for binding to a first epitope of afirst cell-surface molecule, the first proteinaceous molecule providedwith at least one saponin covalently bound via at least one linkerand/or via an oligomeric or polymeric scaffold to an amino-acid residueof said first proteinaceous molecule, or covalently bound directly to anamino-acid residue of said first proteinaceous molecule.

An embodiment within the second series of aspects and embodiments of theinvention is the first proteinaceous molecule according to theinvention, wherein the first binding site comprises or consists of animmunoglobulin, or at least one binding domain of an immunoglobulinand/or at least one binding fragment of an immunoglobulin, such as anantibody, an IgG, a molecule comprising or consisting of a Vhh domain orVh domain, a Fab, an scFv, an Fv, a dAb, an F(ab)₂, Fcab fragment,and/or comprises or consists of at least one ligand for binding to acell-surface molecule such as EGF or a cytokine.

An embodiment within the second series of aspects and embodiments of theinvention is the first proteinaceous molecule according to theinvention, wherein the first epitope of the first cell-surface moleculeis a tumor-cell specific first epitope of a first tumor-cell surfacemolecule, more preferably a tumor-cell specific first epitope of a firsttumor-cell surface receptor specifically present on a tumor cell.

An embodiment within the second series of aspects and embodiments of theinvention is the first proteinaceous molecule according to theinvention, wherein the at least one saponin is a triterpenoid saponinand/or a bisdesmosidic triterpene saponin belonging to the type of a12,13-dehydrooleanane with an aldehyde function in position C-23 andoptionally comprising a glucuronic acid function in a carbohydratesubstituent at the C-3beta-OH group of the saponin, and/or a saponinisolated from a Gypsophila species and/or a Saponaria species and/or anAgrostemma species and/or a Quillaja species such as Quillaja saponaria.

An embodiment within the second series of aspects and embodiments of theinvention is the first proteinaceous molecule according to theinvention, wherein the at least one saponin is a single specific saponinor is a mixture of two or more different saponins, such as one or moreof the saponins in Table A1 or Scheme I, SO1861, SA1657, GE1741, SA1641,QS-21, QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B, QS-21 B-api, QS-21B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862,Quillajasaponin, Saponinum album, QS-18, Quil-A, Gyp1, gypsoside A, AG1,AG2, SO1542, SO1584, SO1658, SO1674, SO1832, or any of their stereomersand/or any combinations thereof, preferably the saponin is SO1861 and/orGE1741 and/or SA1641 and/or QS-21 and/or saponin with a quillaic acidaglycon core, a Gal-(1→2)-[Xyl-(1→3)]-GlcA carbohydrate substituent atthe C-3beta-OH group and aGlc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuccarbohydrate substituent at the C-28-OH group, and/or is3-O-beta-D-galactopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)]-beta-D-glucuronopyranosylquillaic acid28-O-beta-D-glucopyranosyl-(1→3)-beta-D-xylopyranosyl-(1→4)-alpha-L-rhamnopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)-4OAc-beta-D-quinovopyranosyl-(1→4)]-beta-D-fucopyranoside,more preferably the saponin is SO1861 and/or QS-21.

An embodiment within the second series of aspects and embodiments of theinvention is the first proteinaceous molecule according to theinvention, wherein the at least one saponin is a bisdesmosidic saponinhaving a molecular mass of at least 1.500 Dalton and comprising anoleanan-type triterpene containing an aldehyde group at the C-23position and optionally a hydroxyl group at the C-16 position, with afirst branched carbohydrate side chain at the C-3 position which firstbranched carbohydrate side chain optionally contains glucuronic acid,wherein the saponin contains an ester group with a second branchedcarbohydrate side chain at the C-28 position which second branchedcarbohydrate chain preferably comprises at least four carbohydrateunits, optionally containing at least one acetyl residue such as twoacetyl residues and/or optionally comprising deoxy carbohydrates and/oroptionally comprising quinovose and/or optionally comprising glucoseand/or optionally comprising 4-methoxycinnamic acid and/or optionallycomprising5-O-[5-O-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoicacid and/or optionally comprising5-O-[5-O-Rha-(1→2)-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoicacid bound to a carbohydrate via an ester bond, or wherein the at leastone saponin is QS-21 or any one or more of QS-21A, QS-21 A-api, QS-21A-xyl, QS-21B, QS-21 B-api, QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api,QS-17-xyl, QS-18, QS1861, protonated QS1861 (QS1862), Quil-A.

An embodiment within the second series of aspects and embodiments of theinvention is the first proteinaceous molecule according to theinvention, wherein the at least one saponin is a bisdesmosidictriterpene saponin belonging to the type of a 12,13-dehydrooleanane withan aldehyde function in position C-23, wherein the at least one saponinis covalently coupled to the amino-acid residue of the firstproteinaceous molecule via an aldehyde function in the saponin,preferably said aldehyde function in position C-23, preferably via atleast one linker, more preferably via at least one cleavable linker,wherein the amino-acid residue preferably is selected from cysteine andlysine.

An embodiment is the first proteinaceous molecule according to theinvention, wherein the at least one saponin is a bisdesmosidictriterpene saponin belonging to the type of a 12,13-dehydrooleanane withan aldehyde function in position C-23 and comprising a glucuronic acidfunction in a carbohydrate substituent at the C-3beta-OH group of thesaponin, wherein the at least one saponin is covalently coupled to theamino-acid residue of the first proteinaceous molecule via theglucuronic acid function in the carbohydrate substituent at theC-3beta-OH group of the saponin, preferably via at least one linker,wherein the amino-acid residue preferably is selected from cysteine andlysine.

An embodiment within the second series of aspects and embodiments of theinvention is the first proteinaceous molecule according to theinvention, wherein the aldehyde function in position C-23 of the atleast one saponin is covalently coupled to linker N-ε-maleimidocaproicacid hydrazide, which linker is covalently coupled via a thio-ether bondto a sulfhydryl group in the first proteinaceous molecule, such as asulfhydryl group of a cysteine.

An embodiment within the second series of aspects and embodiments of theinvention is the first proteinaceous molecule according to theinvention, wherein the glucuronic acid function in the carbohydratesubstituent at the C-3beta-OH group of the at least one saponin iscovalently coupled to linker1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate, which linker is covalently coupled via anamide bond to an amine group in the first proteinaceous molecule, suchas an amine group of a lysine or an N-terminus of the firstproteinaceous molecule.

An embodiment within the second series of aspects and embodiments of theinvention is the first proteinaceous molecule according to theinvention, wherein the first epitope of the first cell-surface moleculeto which the first binding site of the first proteinaceous moleculebinds is a tumor-cell specific first epitope of the tumor-cell specificreceptor preferably selected from CD71, CA125, EpCAM(17-1A), CD52, CEA,CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrin alpha-Vbeta-3, HER2, EGFR, CD20, CD22, Folate receptor 1, CD146, CD56, CD19,CD138, CD27L receptor, PSMA, CanAg, integrin-alphaV, CA6, CD33,mesothelin, Cripto, CD3, CD30, CD239, CD70, CD123, CD352, DLL3, CD25,ephrinA4, MUC1, Trop2, CEACAM5, CEACAM6, HER3, CD74, PTK7, Notch3, FGF2,C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA4, CD52, PDGFRA, VEGFR1,VEGFR2, more preferably selected from CD71, EGFR, HER2.

An embodiment within the second series of aspects and embodiments of theinvention is the first proteinaceous molecule according to theinvention, wherein the tumor cell-specific first epitope, firsttumor-cell surface molecule or first tumor-cell specific receptor, are afirst epitope or a first molecule or a first receptor that areinternalized by the tumor cell after binding of the first proteinaceousmolecule of the invention to the first epitope or first molecule orfirst receptor, and wherein preferably the first proteinaceous moleculeis subjected to tumor-cell receptor-mediated internalization, e.g. viaendocytosis, or tumor-cell surface molecule mediated internalization,e.g. via endocytosis, when bound to the cell-surface molecule comprisingthe first epitope, the tumor-cell surface molecule or the tumor-cellspecific receptor.

An embodiment within the second series of aspects and embodiments of theinvention is the first proteinaceous molecule according to theinvention, wherein the first binding site of the first proteinaceousmolecule comprises or consists of any one of cetuximab, daratumumab,gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab,moxetumomab, polatuzumab, obinutuzumab, OKT-9 anti-CD71 monoclonalantibody of the IgG type, pertuzumab, rituximab, ofatumumab, Herceptin,alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal antibody, anantibody of Table A2 or Table A3 or Table A4, preferably cetuximab ortrastuzumab or OKT-9, or at least one tumor-cell receptorbinding-fragment thereof and/or at least one tumor-cell receptorbinding-domain thereof, preferably at least one tumor-cell specificreceptor binding-fragment thereof and/or at least one tumor-cellspecific receptor binding-domain thereof.

An aspect of the invention within the second series of aspects andembodiments of the invention relates to a therapeutic combination,wherein the therapeutic combination comprises: (a) a firstpharmaceutical composition comprising the first proteinaceous moleculeaccording to the invention and optionally a pharmaceutically acceptableexcipient; and (b) a second pharmaceutical composition comprising asecond proteinaceous molecule different from the first proteinaceousmolecule, the second proteinaceous molecule comprising a second bindingsite for binding to a second epitope of a second cell-surface moleculedifferent from the first cell-surface molecule, and comprising aneffector moiety, the second pharmaceutical composition optionallyfurther comprising a pharmaceutically acceptable excipient, wherein thesecond epitope is different from the first epitope.

An embodiment within the second series of aspects and embodiments of theinvention is the therapeutic combination according to the invention,wherein the therapeutic combination comprises: (a) the firstpharmaceutical composition according to the invention comprising thefirst proteinaceous molecule according to the invention, wherein thefirst epitope on the first cell-surface molecule is a tumor-cellspecific first epitope on a first tumor cell-specific surface molecule,preferably a tumor-cell specific first epitope on a first cell-surfacereceptor specifically present at a tumor cell; and (b) the secondpharmaceutical composition according to the invention, wherein thesecond cell-surface molecule is a second tumor cell-specific surfacemolecule different from the first tumor cell-specific surface molecule,preferably a second cell-surface receptor specifically present at atumor cell different from the first cell-surface receptor specificallypresent at said tumor cell, and wherein the second epitope is atumor-cell specific second epitope.

An embodiment within the second series of aspects and embodiments of theinvention is the therapeutic combination according to the invention,wherein the therapeutic combination comprises: (a) the firstpharmaceutical composition according to the invention comprising thefirst proteinaceous molecule according to the invention and comprisingthe first binding site for binding to the first epitope on the firstcell-surface molecule, the first pharmaceutical composition optionallyfurther comprising a pharmaceutically acceptable excipient; and (b) athird pharmaceutical composition comprising a third proteinaceousmolecule, the third proteinaceous molecule comprising the first bindingsite for binding to the first epitope on the cell-surface molecule of(a) and an effector moiety, the third pharmaceutical compositionoptionally further comprising a pharmaceutically acceptable excipient,wherein the first binding site of the first proteinaceous molecule andthe first binding site of the third proteinaceous molecule are the same,and wherein the first cell-surface molecule and the first epitope on thefirst cell-surface molecule, to which the first proteinaceous moleculecan bind, and the first cell-surface molecule and the first epitope onthe first cell-surface molecule, to which the third proteinaceousmolecule can bind, are the same.

An embodiment within the second series of aspects and embodiments of theinvention is the therapeutic combination according to the invention,wherein the therapeutic combination comprises: (a) the firstpharmaceutical composition according to the invention; and (b) the thirdpharmaceutical composition according to the invention, wherein the firstcell-surface molecule is expressed on a tumor cell surface, andpreferably the first cell-surface molecule is a tumor cell-specificsurface molecule, and wherein preferably the first epitope is a firsttumor-cell specific epitope.

An embodiment within the second series of aspects and embodiments of theinvention is the first proteinaceous molecule according to the inventionor the therapeutic combination according to the invention, wherein thefirst binding site for binding to the first epitope on the first cellsurface molecule is a binding site for a tumor-cell specific firstepitope on a first cell-surface receptor specifically present at a tumorcell.

An embodiment within the second series of aspects and embodiments of theinvention is the therapeutic combination according to the invention,wherein the second binding site of the second proteinaceous moleculeand/or the first binding site of the third proteinaceous moleculecomprises or consists of an immunoglobulin, at least one binding domainof an immunoglobulin and/or at least one binding fragment of animmunoglobulin, such as an antibody, an IgG, a molecule comprising orconsisting of a Vhh domain or Vh domain, a Fab, an scFv, an Fv, a dAb,an F(ab)2, Fcab fragment, and/or comprises or consists of at least oneligand for binding to a cell-surface molecule such as EGF or a cytokine.

An embodiment within the second series of aspects and embodiments of theinvention is the therapeutic combination according to the invention,wherein the second binding site of the second proteinaceous molecule forbinding to the second epitope is a second binding site for a tumor-cellspecific second epitope on a second cell-surface receptor specificallypresent at the tumor cell, wherein the second binding site is differentfrom the first binding site.

An embodiment within the second series of aspects and embodiments of theinvention is the first proteinaceous molecule according to the inventionor the therapeutic combination according to the invention, wherein saidfirst and second proteinaceous molecules comprise the first and secondbinding site respectively for binding to a first and a second tumor-cellspecific epitope on a first and a second tumor-cell specific receptorrespectively, the receptors being different and being present at thesame tumor cell, wherein the first and second binding site are differentand the first and second tumor cell specific epitope are different.

An embodiment within the second series of aspects and embodiments of theinvention is the first proteinaceous molecule according to the inventionor the therapeutic combination according to the invention, wherein saidfirst and third proteinaceous molecules comprise the same first bindingsite for binding to a first tumor-cell specific epitope on a firsttumor-cell specific receptor.

An embodiment within the second series of aspects and embodiments of theinvention is the first proteinaceous molecule according to the inventionor the therapeutic combination according to the invention wherein thefirst receptor and/or the second receptor are selected from CD71, CA125,EpCAM(17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1,vascular integrin alpha-V beta-3, HER2, EGFR, CD20, CD22, Folatereceptor 1, CD146, CD56, CD19, CD138, CD27L receptor, PSMA, CanAg,integrin-alphaV, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239, CD70,CD123, CD352, DLL3, CD25, ephrinA4, MUC1, Trop2, CEACAM5, CEACAM6, HER3,CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA4,CD52, PDGFRA, VEGFR1, VEGFR2, preferably selected from CD71, EGFR andHER2.

An embodiment within the second series of aspects and embodiments of theinvention is the first proteinaceous molecule according to the inventionand/or the therapeutic combination according to the invention, whereinthe first and second tumor-cell specific receptors are internalized bythe tumor cell after binding to the first proteinaceous moleculeaccording to the invention and/or the second proteinaceous moleculeaccording to the invention, and wherein preferably binding of the firstproteinaceous molecule and/or the second proteinaceous molecule to thefirst and second tumor-cell specific receptors respectively, results intumor-cell receptor-mediated internalization, e.g. via endocytosis, of acomplex of the first proteinaceous molecule and the first tumor-cellspecific receptor and of a complex of the second proteinaceous moleculeand the second tumor-cell specific receptor.

An embodiment within the second series of aspects and embodiments of theinvention is the therapeutic combination or the first pharmaceuticalcomposition according the invention, wherein the first tumor-cellreceptor, preferably the first tumor-cell specific receptor, isinternalized by the tumor cell after binding to the first proteinaceousmolecule according to the invention and/or after binding to the thirdproteinaceous molecule according to the invention, and whereinpreferably binding of the first proteinaceous molecule and/or the thirdproteinaceous molecule to the first tumor-cell receptor, such as thefirst tumor-cell specific receptor, is followed by tumor-cellreceptor-mediated internalization, e.g. via endocytosis, of a complex ofthe first proteinaceous molecule and the first tumor-cell receptor andof a complex of the third proteinaceous molecule and the firsttumor-cell receptor.

An embodiment within the second series of aspects and embodiments of theinvention is the first proteinaceous molecule according to theinvention, and/or therapeutic combination according to the invention,wherein the first binding site and/or the second binding site is/are orcomprise(s) a monoclonal antibody or at least one cell-surface moleculebinding fragment and/or -domain thereof, and preferably comprise orconsist of any one of cetuximab, daratumumab, gemtuzumab, trastuzumab,panitumumab, brentuximab, inotuzumab, moxetumomab, polatuzumab,obinutuzumab, OKT-9 anti-CD71 monoclonal antibody of the IgG type,pertuzumab, rituximab, ofatumumab, Herceptin, alemtuzumab, pinatuzumab,OKT-10 anti-CD38 monoclonal antibody, and an antibody of Table A4,preferably cetuximab or trastuzumab or OKT-9, or at least onecell-surface molecule binding fragment or -domain thereof, with theproviso that the first binding site of the first proteinaceous moleculeis different from the second binding site of the second proteinaceousmolecule.

An embodiment within the second series of aspects and embodiments of theinvention is the therapeutic combination according to the invention orthe first pharmaceutical composition according to the invention, whereinthe first binding site of the first proteinaceous molecule and the thirdproteinaceous molecule comprises a monoclonal antibody or at least oneof a cell-surface molecule binding domain and/or -fragment thereof, andpreferably comprise or consist of any one of cetuximab, daratumumab,gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab,moxetumomab, polatuzumab, obinutuzumab, OKT-9 anti-CD71 monoclonalantibody of the IgG type, pertuzumab, rituximab, ofatumumab, Herceptin,alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal antibody, anantibody of Table A2 or Table A3 or Table A4, preferably cetuximab ortrastuzumab or OKT-9, or at least one cell-surface molecule bindingfragment and/or -domain thereof, with the proviso that the first bindingsite of the first proteinaceous molecule is the same as the firstbinding site of the third proteinaceous molecule.

An embodiment within the second series of aspects and embodiments of theinvention is the therapeutic combination according to the invention,wherein the second binding site of the second proteinaceous moleculeand/or the first binding site of the third proteinaceous molecule is orcomprises a monoclonal antibody or at least one cell-surface moleculebinding fragment or -domain thereof, and preferably comprises orconsists of any one of Gemtuzumab ozogamicin, Brentuximab vedotin,Trastuzumab emtansine, Inotuzumab ozogamicin, Moxetumomab pasudotox andPolatuzumab vedotin and an antibody-drug conjugate of Table A2 and TableA3.

An embodiment within the second series of aspects and embodiments of theinvention is the therapeutic combination according to the invention,wherein the effector moiety that is comprised by the secondproteinaceous molecule and/or by the third proteinaceous moleculecomprises or consists of any one or more of an oligonucleotide, anucleic acid, a xeno nucleic acid, preferably selected from any one ormore of a vector, a gene, a cell suicide inducing transgene,deoxyribonucleic acid (DNA), ribonucleic acid (RNA), anti-senseoligonucleotide (ASO, AON), short interfering RNA (siRNA), microRNA(miRNA), DNA aptamer, RNA aptamer, mRNA, mini-circle DNA, peptidenucleic acid (PNA), phosphoramidate morpholino oligomer (PMO), lockednucleic acid (LNA), bridged nucleic acid (BNA),2′-deoxy-2′-fluoroarabino nucleic acid (FANA), 2′-O-methoxyethyl-RNA(MOE), 2′-0,4′-aminoethylene bridged nucleic acid, 3′-fluoro hexitolnucleic acid (FHNA), a plasmid, glycol nucleic acid (GNA) and threosenucleic acid (TNA), or a derivative thereof, more preferably a BNA, forexample a BNA for silencing HSP27 protein expression.

An embodiment within the second series of aspects and embodiments of theinvention is the therapeutic combination according to the invention,wherein the effector moiety that is comprised by the secondproteinaceous molecule and/or by the third proteinaceous moleculecomprises or consists of at least one proteinaceous molecule, preferablyselected from any one or more of a peptide, a protein, an enzyme such asurease and Cre-recombinase, a ribosome-inactivating protein, aproteinaceous toxin, more preferably selected from any one or more of aprotein toxin selected from Table A5 and/or a viral toxin such asapoptin; a bacterial toxin such as Shiga toxin, Shiga-like toxin,Pseudomonas aeruginosa exotoxin (PE) or exotoxin A of PE, full-length ortruncated diphtheria toxin (DT), cholera toxin; a fungal toxin such asalpha-sarcin; a plant toxin including ribosome-inactivating proteins andthe A chain of type 2 ribosome-inactivating proteins such as dianthine.g. dianthin-30 or dianthin-32, saporin e.g. saporin-S3 or saporin-S6,bouganin or de-immunized derivative debouganin of bouganin, shiga-liketoxin A, pokeweed antiviral protein, ricin, ricin A chain, modeccin,modeccin A chain, abrin, abrin A chain, volkensin, volkensin A chain,viscumin, viscumin A chain; or an animal or human toxin such as frogRNase, or granzyme B or angiogenin from humans, or any fragment orderivative thereof; preferably the protein toxin is dianthin and/orsaporin.

An embodiment within the second series of aspects and embodiments of theinvention is the therapeutic combination according to the invention,wherein the effector moiety comprised by the second proteinaceousmolecule and/or by the third proteinaceous molecule comprises orconsists of at least one payload, preferably selected from any one ormore of a toxin targeting ribosomes, a toxin targeting elongationfactors, a toxin targeting tubulin, a toxin targeting DNA and a toxintargeting RNA, more preferably any one or more of emtansine, pasudotox,maytansinoid derivative DM1, maytansinoid derivative DM4, monomethylauristatin E (MMAE, vedotin), monomethyl auristatin F (MMAF, mafodotin),a Calicheamicin, N-Acetyl-γ-calicheamicin, a pyrrolobenzodiazepine (PBD)dimer, a benzodiazepine, a CC-1065 analogue, a duocarmycin, Doxorubicin,paclitaxel, docetaxel, cisplatin, cyclophosphamide, etoposide,docetaxel, 5-fluorouracyl (5-FU), mitoxantrone, a tubulysin, anindolinobenzodiazepine, AZ13599185, a cryptophycin, rhizoxin,methotrexate, an anthracycline, a camptothecin analogue, SN-38,DX-8951f, exatecan mesylate, truncated form of Pseudomonas aeruginosaexotoxin (PE38), a Duocarmycin derivative, an amanitin, a-amanitin, aspliceostatin, a thailanstatin, ozogamicin, tesirine, Amberstatin269 andsoravtansine, or a derivative thereof.

An embodiment within the second series of aspects and embodiments of theinvention is the first proteinaceous molecule according to theinvention, wherein the first proteinaceous molecule comprises more thanone saponin, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32, 64 or 1-100saponins, or any number of saponins therein between, such as 7, 9, 12saponins, covalently bound directly to an amino-acid residue of thefirst proteinaceous molecule, preferably to a cysteine and/or to alysine, and/or covalently bound via at least one linker and/or via atleast one cleavable linker and/or via at least one polymeric oroligomeric scaffold, preferably 1-8 of such scaffolds or 2-4 of suchscaffolds, wherein the at least one scaffold is optionally based on adendron, wherein 1-32 saponins such as 2, 3, 4, 5, 6, 8, 10, 16, 32saponins, or any number of saponins therein between, such as 7, 9, 12saponins, are covalently bound to the at least one scaffold.

An embodiment within the second series of aspects and embodiments of theinvention is the first proteinaceous molecule according to theinvention, wherein the at least one linker is a non-cleavable linker ora cleavable linker, wherein the cleavable linker is for example subjectto cleavage under acidic conditions, reductive conditions, enzymaticconditions or light-induced conditions, and preferably the cleavablelinker comprises a hydrazone bond or a hydrazide bond subject tocleavage under acidic conditions when bound to saponin, and/or comprisesa bond susceptible to proteolysis, for example proteolysis by CathepsinB, and/or is a bond susceptible for cleavage under reductive conditionssuch as a disulphide bond, when bound to saponin.

An embodiment within the second series of aspects and embodiments of theinvention is the first proteinaceous molecule according to theinvention, wherein the cleavable linker is subject to cleavage in vivounder acidic conditions as present in endosomes and/or lysosomes ofmammalian cells, preferably human cells, preferably at pH 4.0-6.5, andmore preferably at pH 5.5, when the cleavable linker is bound to asaponin.

An embodiment within the second series of aspects and embodiments of theinvention is the first proteinaceous molecule according to theinvention, wherein the oligomeric or polymeric scaffold comprises apolymeric or oligomeric structure and comprises a chemical group, thechemical group for covalently coupling of the scaffold to the amino-acidresidue of said first proteinaceous molecule.

An embodiment within the second series of aspects and embodiments of theinvention is the first proteinaceous molecule according to theinvention, wherein the at least one saponin is covalently bound to thepolymeric or oligomeric structure of the oligomeric or polymericscaffold via at least one cleavable linker according to the invention.

An embodiment within the second series of aspects and embodiments of theinvention is the first proteinaceous molecule according to theinvention, wherein the chemical group of the oligomeric or polymericscaffold, for covalently coupling of the oligomeric or polymericscaffold to the amino-acid residue of said first proteinaceous molecule,is a click chemistry group, preferably selected from a tetrazine, anazide, an alkene or an alkyne, or a cyclic derivative of these groups,more preferably said chemical group is an azide.

An embodiment within the second series of aspects and embodiments of theinvention is the first proteinaceous molecule according to theinvention, wherein the polymeric or oligomeric structure of theoligomeric or polymeric scaffold comprises a linear, branched and/orcyclic polymer, oligomer, dendrimer, dendron, dendronized polymer,dendronized oligomer, a DNA, a polypeptide, poly-lysine, a poly-ethyleneglycol, or an assembly of these polymeric or oligomeric structures whichassembly is preferably built up by covalent cross-linking.

An aspect of the invention within the second series of aspects andembodiments of the invention relates to a composition comprising thefirst proteinaceous molecule according to the invention and the secondproteinaceous molecule according to the invention.

An aspect of the invention within the second series of aspects andembodiments of the invention relates to a composition comprising thefirst proteinaceous molecule according to the invention and the thirdproteinaceous molecule according to the invention.

An embodiment within the second series of aspects and embodiments of theinvention is the composition according to the invention, wherein theeffector moiety that is comprised by the second proteinaceous moleculeor by the third proteinaceous molecule is any one of the effectormoieties according to the invention, preferably a BNA.

An aspect of the invention within the second series of aspects andembodiments of the invention relates to a composition comprising thefirst proteinaceous molecule according to the invention and any one ormore of an oligonucleotide, a nucleic acid and a xeno nucleic acid,preferably selected from at least one of a vector, a gene, a cellsuicide inducing transgene, deoxyribonucleic acid (DNA), ribonucleicacid (RNA), anti-sense oligonucleotide (ASO, AON), short interfering RNA(siRNA), microRNA (miRNA), DNA aptamer, RNA aptamer, mRNA, mini-circleDNA, peptide nucleic acid (PNA), phosphoramidate morpholino oligomer(PMO), locked nucleic acid (LNA), bridged nucleic acid (BNA),2′-deoxy-2′-fluoroarabino nucleic acid (FANA), 2′-O-methoxyethyl-RNA(MOE), 2′-0,4′-aminoethylene bridged nucleic acid, 3′-fluoro hexitolnucleic acid (FHNA), a plasmid, glycol nucleic acid (GNA) and threosenucleic acid (TNA), or a derivative thereof, more preferably a BNA, forexample a BNA for silencing HSP27 protein expression.

An aspect of the invention within the second series of aspects andembodiments of the invention relates to an antibody-drug conjugate or aligand-drug conjugate comprising the first proteinaceous moleculeaccording to the invention and an effector moiety.

An embodiment within the second series of aspects and embodiments of theinvention is the antibody-drug conjugate or the ligand-drug conjugateaccording to the invention, wherein the antibody can bind to any one ofCD71, CA125, EpCAM(17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin,syndecan-1, vascular integrin alpha-V beta-3, HER2, EGFR, CD20, CD22,Folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, PSMA,CanAg, integrin-alphaV, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239,CD70, CD123, CD352, DLL3, CD25, ephrinA4, MUC1, Trop2, CEACAM5, CEACAM6,HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1,CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2, preferably CD71, HER2, EGFR, and/oris or comprises any one of cetuximab, daratumumab, gemtuzumab,trastuzumab, panitumumab, brentuximab, inotuzumab, moxetumomab,polatuzumab, obinutuzumab, OKT-9 anti-CD71 monoclonal antibody of theIgG type, pertuzumab, rituximab, ofatumumab, Herceptin, alemtuzumab,pinatuzumab, OKT-10 anti-CD38 monoclonal antibody, an antibody of TableA2 or Table A3 or Table A4, preferably cetuximab or trastuzumab orOKT-9, or at least one tumor-cell receptor binding-fragment thereofand/or at least one tumor-cell receptor binding-domain thereof, and/orwherein the antibody-drug conjugate comprises any one of Gemtuzumabozogamicin, Brentuximab vedotin, Trastuzumab emtansine, Inotuzumabozogamicin, Moxetumomab pasudotox and Polatuzumab vedotin and anantibody-drug conjugate of Table A2 and Table A3, or wherein theligand-drug conjugate comprises at least one ligand for binding to acell-surface molecule such as EGF or a cytokine.

An embodiment within the second series of aspects and embodiments of theinvention is the antibody-drug conjugate or the ligand-drug conjugateaccording to the invention, wherein the effector moiety is any one ormore of the effector moieties according to the invention.

An aspect of the invention within the second series of aspects andembodiments of the invention relates to a pharmaceutical compositioncomprising the composition according to the invention or theantibody-drug conjugate according to the invention or the ligand-drugconjugate according to the invention, and optionally further comprisinga pharmaceutically acceptable excipient.

An aspect of the invention within the second series of aspects andembodiments of the invention relates to the therapeutic combination orthe composition or the antibody-drug conjugate or the ligand-drugconjugate or the pharmaceutical composition according to the invention,for use as a medicament.

An aspect of the invention within the second series of aspects andembodiments of the invention relates to the therapeutic combination orthe composition or the antibody-drug conjugate or the ligand-drugconjugate or the pharmaceutical composition according to the invention,for use in the treatment or prevention of a cancer or an autoimmunedisease.

An aspect of the invention within a third series of aspects andembodiments of the invention relates to an effector moiety capable ofinducing an intracellular effect when present inside a mammalian cell,the effector moiety conjugated with at least one saponin, wherein the atleast one saponin is covalently bound to the effector moiety via atleast one linker, or is covalently bound directly to said effectormoiety.

An embodiment within the third series of aspects and embodiments of theinvention is the effector moiety according to the invention, comprisingor consisting of at least one oligonucleotide, a nucleic acid, a xenonucleic acid, preferably selected from any one or more of a vector, agene, a cell suicide inducing transgene, deoxyribonucleic acid (DNA),ribonucleic acid (RNA), anti-sense oligonucleotide (ASO, AON), shortinterfering RNA (siRNA), microRNA (miRNA), DNA aptamer, RNA aptamer,mRNA, mini-circle DNA, peptide nucleic acid (PNA), phosphoramidatemorpholino oligomer (PMO), locked nucleic acid (LNA), bridged nucleicacid (BNA), 2′-deoxy-2′-fluoroarabino nucleic acid (FANA),2′-O-methoxyethyl-RNA (MOE), 2′-0,4′-aminoethylene bridged nucleic acid,3′-fluoro hexitol nucleic acid (FHNA), a plasmid, glycol nucleic acid(GNA) and threose nucleic acid (TNA), or a derivative thereof, morepreferably a BNA, for example a BNA for silencing HSP27 proteinexpression.

An embodiment within the third series of aspects and embodiments of theinvention is the effector moiety according to the invention, comprisingat least one proteinaceous molecule, the proteinaceous moleculepreferably selected from any one or more of a peptide, a protein, anenzyme such as urease and Cre-recombinase, a ribosome-inactivatingprotein, a proteinaceous toxin such as any one or more of a proteintoxin selected from Table A5 and/or a bacterial toxin or plant toxin,more preferably selected from any one or more of a viral toxin such asapoptin; a bacterial toxin such as Shiga toxin, Shiga-like toxin,Pseudomonas aeruginosa exotoxin (PE) or exotoxin A of PE, full-length ortruncated diphtheria toxin (DT), cholera toxin; a fungal toxin such asalpha-sarcin; a plant toxin including ribosome-inactivating proteins andthe A chain of type 2 ribosome-inactivating proteins such as dianthine.g. dianthin-30 or dianthin-32, saporin e.g. saporin-S3 or saporin-S6,bouganin or de-immunized derivative debouganin of bouganin, shiga-liketoxin A, pokeweed antiviral protein, ricin, ricin A chain, modeccin,modeccin A chain, abrin, abrin A chain, volkensin, volkensin A chain,viscumin, viscumin A chain; or an animal or human toxin such as frogRNase, or granzyme B or angiogenin from humans, or any fragment orderivative thereof; preferably the protein toxin is dianthin and/orsaporin.

An embodiment within the third series of aspects and embodiments of theinvention is the effector moiety according to the invention, comprisingat least one payload, the payload preferably selected from any one ormore of a toxin targeting ribosomes, a toxin targeting elongationfactors, a toxin targeting tubulin, a toxin targeting DNA and a toxintargeting RNA, more preferably any one or more of emtansine, pasudotox,maytansinoid derivative DM1, maytansinoid derivative DM4, monomethylauristatin E (MMAE, vedotin), monomethyl auristatin F (MMAF, mafodotin),a Calicheamicin, N-Acetyl-γ-calicheamicin, a pyrrolobenzodiazepine (PBD)dimer, a benzodiazepine, a CC-1065 analogue, a duocarmycin, Doxorubicin,paclitaxel, docetaxel, cisplatin, cyclophosphamide, etoposide,docetaxel, 5-fluorouracyl (5-FU), mitoxantrone, a tubulysin, anindolinobenzodiazepine, AZ13599185, a cryptophycin, rhizoxin,methotrexate, an anthracycline, a camptothecin analogue, SN-38,DX-8951f, exatecan mesylate, truncated form of Pseudomonas aeruginosaexotoxin (PE38), a Duocarmycin derivative, an amanitin, a-amanitin, aspliceostatin, a thailanstatin, ozogamicin, tesirine, Amberstatin269 andsoravtansine, or a derivative thereof.

An embodiment within the third series of aspects and embodiments of theinvention is the effector moiety according to the invention, wherein theat least one saponin is a triterpenoid saponin and/or a bisdesmosidictriterpene saponin belonging to the type of a 12,13-dehydrooleanane withan aldehyde function in position C-23 and optionally comprising aglucuronic acid function in a carbohydrate substituent at the C-3beta-OHgroup of the saponin, and/or a saponin isolated from a Gypsophilaspecies and/or a Saponaria species and/or an Agrostemma species and/or aQuillaja species such as Quillaja saponaria.

An embodiment within the third series of aspects and embodiments of theinvention is the effector moiety according to the invention, wherein theat least one saponin is a single specific saponin or is a mixture of twoor more different saponins.

An embodiment within the third series of aspects and embodiments of theinvention is the effector moiety according to the invention, wherein thesaponin is one or more of the saponins in Table A1 or Scheme I, SO1861,SA1657, GE1741, SA1641, QS-21, QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B,QS-21 B-api, QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl,QS1861, QS1862, Quillajasaponin, Saponinum album, QS-18, Quil-A, Gyp1,gypsoside A, AG1, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, or any oftheir stereomers and/or any combinations thereof, preferably the saponinis SO1861 and/or GE1741 and/or SA1641 and/or QS-21 and/or saponin with aquillaic acid aglycon core, a Gal-(1→2)-[Xyl-(1→3)]-GlcA carbohydratesubstituent at the C-3beta-OH group and aGlc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuccarbohydrate substituent at the C-28-OH group, and/or is3-O-beta-D-galactopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)]-beta-D-glucuronopyranosylquillaic acid28-O-beta-D-glucopyranosyl-(1→3)-beta-D-xylopyranosyl-(1→4)-alpha-L-rhamnopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)-4-OAc-beta-D-quinovopyranosyl-(1→4)]-beta-D-fucopyranoside,more preferably the saponin is SO1861 and/or QS-21.

An embodiment within the third series of aspects and embodiments of theinvention is the effector moiety according to the invention, wherein thesaponin is a bisdesmosidic saponin having a molecular mass of at least1.500 Dalton and comprising an oleanan-type triterpene containing analdehyde group at the C-23 position and optionally a hydroxyl group atthe C-16 position, with a first branched carbohydrate side chain at theC-3 position which first branched carbohydrate side chain optionallycontains glucuronic acid, wherein the saponin contains an ester groupwith a second branched carbohydrate side chain at the C-28 positionwhich second branched carbohydrate chain preferably comprises at leastfour carbohydrate units and optionally contains at least one acetylresidue such as two acetyl residues and/or optionally comprises one ormore deoxy carbohydrates and/or quinovose and/or glucose and/or4-methoxycinnamic acid and/or optionally comprising5-O-[5-O-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoicacid and/or optionally comprising5-O-[5-O-Rha-(1→2)-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoicacid bound to a carbohydrate via an ester bond, or wherein the at leastone saponin is QS-21 or any one or more of QS-21A, QS-21 A-api, QS-21A-xyl, QS-21B, QS-21 B-api, QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api,QS-17-xyl, QS-18, QS1861, protonated QS1861 (QS1862), Quil-A.

An embodiment within the third series of aspects and embodiments of theinvention is the effector moiety according to the invention, wherein theat least one saponin is a bisdesmosidic triterpene saponin belonging tothe type of a 12,13-dehydrooleanane with an aldehyde function inposition C-23, wherein the at least one saponin is covalently coupled toan amino-acid residue, when present, of the effector moiety via analdehyde function in the saponin, preferably said aldehyde function inposition C-23, preferably via at least one linker, more preferably viaat least one cleavable linker, wherein the amino-acid residue preferablyis selected from cysteine and lysine.

An embodiment within the third series of aspects and embodiments of theinvention is the effector moiety according to the invention, wherein theat least one saponin is a bisdesmosidic triterpene saponin belonging tothe type of a 12,13-dehydrooleanane with an aldehyde function inposition C-23 and comprising a glucuronic acid function in acarbohydrate substituent at the C-3beta-OH group of the saponin, whereinthe at least one saponin is covalently coupled to an amino-acid residue,when present, of the effector moiety via the glucuronic acid function inthe carbohydrate substituent at the C-3beta-OH group of the saponin,preferably via at least one linker, wherein the amino-acid residuepreferably is selected from cysteine and lysine.

An embodiment within the third series of aspects and embodiments of theinvention is the effector moiety according to the invention, wherein theat least one linker comprises at least one non-cleavable linker and/orat least one cleavable linker, wherein optionally said cleavable linkeris subject to cleavage under acidic, reductive, enzymatic orlight-induced conditions, and preferably the cleavable linker comprisesa cleavable bond selected from a hydrazone bond and a hydrazide bondsubject to cleavage under acidic conditions, and/or a bond susceptibleto proteolysis, for example proteolysis by Cathepsin B, and/or a bondsusceptible for cleavage under reductive conditions such as a disulphidebond.

An embodiment within the third series of aspects and embodiments of theinvention is the effector moiety according to the invention, wherein theat least one linker comprises at least one cleavable linker which issubject to cleavage in vivo under acidic conditions as present inendosomes and/or in lysosomes of mammalian cells, preferably of humancells, preferably at pH 4.0-6.5, and more preferably at pH≤5.5.

An embodiment within the third series of aspects and embodiments of theinvention is the effector moiety according to the invention, wherein theat least one saponin is covalently bound to a lysine side chain, formingan amide bond, and/or to a cysteine side chain, forming a thio-etherlinkage, or a disulphide bond, wherein the lysine and/or cysteine is/arecomprised by the effector moiety, and wherein the saponin is boundeither directly to the effector moiety, or bound via at least onelinker.

An embodiment within the third series of aspects and embodiments of theinvention is the effector moiety according to the invention, wherein theat least one saponin is covalently bound to the effector moiety via atleast one linker, wherein the linker is or comprises a scaffoldcomprising a polymeric or oligomeric structure and a chemical group forcovalently coupling of the scaffold to the effector moiety.

An embodiment within the third series of aspects and embodiments of theinvention is the effector moiety according to the invention, wherein theat least one saponin is covalently bound to the polymeric or oligomericstructure of the scaffold via a cleavable bond.

An embodiment within the third series of aspects and embodiments of theinvention is the effector moiety according to the invention, wherein thecleavable bond is subject to cleavage under any of acidic conditions,reductive conditions, enzymatic conditions and light-induced conditions,more preferably the cleavable bond is a hydrazone bond or a hydrazidebond subject to cleavage under acidic conditions, and/or is a bondsusceptible to proteolysis, for example proteolysis by Cathepsin B,and/or is a bond susceptible for cleavage under reductive conditionssuch as a disulphide bond.

An embodiment within the third series of aspects and embodiments of theinvention is the effector moiety according to the invention, wherein thecleavable bond is subject to cleavage in vivo under acidic conditions aspresent in endosomes and/or in lysosomes of mammalian cells, preferablyof human cells, preferably at pH 4.0-6.5, and more preferably at pH≤5.5.

An embodiment within the third series of aspects and embodiments of theinvention is the effector moiety according to the invention, wherein theat least one saponin is covalently bound to the polymeric or oligomericstructure of the scaffold via an imine bond, a hydrazone bond, ahydrazide bond, an oxime bond, a 1,3-dioxolane bond, a disulphide bond,a thio-ether bond, an amide bond, a peptide bond and/or an ester bond,preferably via at least one linker, preferably an amide bond, ahydrazide bond, a thio-ether bond and/or a hydrazone bond.

An embodiment within the third series of aspects and embodiments of theinvention is the effector moiety according to the invention, wherein theat least one saponin is covalently bound to the polymeric or oligomericstructure of the scaffold, involving in the covalent bond the aldehydefunction in position C-23 of the at least one saponin, when present, thecovalent bond being preferably an imine bond or a hydrazone bond or anamide bond or a thio-ether bond or a disulphide bond, and/or involvingin the covalent bond the glucuronic acid function in the carbohydratesubstituent at the C-3beta-OH group of the at least one saponin, whenpresent, wherein preferably the covalent bond is an amide bond or adisulphide bond or a thio-ether bond.

An embodiment within the third series of aspects and embodiments of theinvention is the effector moiety according to the invention, wherein thealdehyde function in position C-23 of the at least one saponin iscovalently coupled to linker N-ε-maleimidocaproic acid hydrazide, whichlinker is covalently coupled via a thio-ether bond to a sulfhydryl groupin the polymeric or oligomeric structure of the scaffold, such as asulfhydryl group of a cysteine.

An embodiment within the third series of aspects and embodiments of theinvention is the effector moiety according to the invention, wherein theglucuronic acid function in the carbohydrate substituent at theC-3beta-OH group of the at least one saponin is covalently coupled tolinker1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate, which linker is covalently coupled via anamide bond to an amine group in the polymeric or oligomeric structure ofthe scaffold, such as an amine group of a lysine or an N-terminus of thepolymeric or oligomeric structure of the scaffold.

An embodiment within the third series of aspects and embodiments of theinvention is the effector moiety according to the invention, wherein thechemical group of the scaffold, for covalently coupling of the scaffoldto the effector moiety, is a click chemistry group, preferably selectedfrom a tetrazine, an azide, an alkene or an alkyne, or a cyclicderivative of these groups, more preferably the click chemistry group isan azide.

An embodiment within the third series of aspects and embodiments of theinvention is the effector moiety according to the invention, wherein thepolymeric or oligomeric structure of the scaffold comprises a linear,branched and/or cyclic polymer, oligomer, dendrimer, dendron,dendronized polymer, dendronized oligomer, a DNA, a polypeptide,poly-lysine, a poly-ethylene glycol, or an assembly of these polymericor oligomeric structures which assembly is preferably built up bycovalent cross-linking.

An embodiment within the third series of aspects and embodiments of theinvention is the effector moiety according to the invention, wherein theat least one saponin is a defined number of saponins or a defined rangeof saponins, preferably 1-128 saponins or at least 2, 3, 4, 5, 6, 8, 10,16, 32, 64 or 128 saponins, or any number of saponins therein between,such as 7, 9, 12 saponins.

An embodiment within the third series of aspects and embodiments of theinvention is the effector moiety according to the invention, wherein theeffector moiety comprises more than one saponin, preferably 2, 3, 4, 5,6, 8, 10, 16, 32, 64 or 1-100 saponins, or any number of saponinstherein between, such as 7, 9, 12 saponins, covalently bound directly toan amino-acid residue of the effector moiety, preferably to a cysteineand/or to a lysine, and/or covalently bound via at least one linkerand/or via at least one cleavable linker and/or via at least onepolymeric or oligomeric scaffold of any one of the claims 14-23,preferably 1-8 of such scaffolds or 2-4 of such scaffolds, wherein 1-32saponins, preferably 2, 3, 4, 5, 6, 8, 10, 16 or 32 saponins, arecovalently bound to the at least one scaffold.

An aspect of the invention within the third series of aspects andembodiments of the invention relates to an antibody-drug conjugatecomprising the effector moiety according to the invention, or aligand-drug conjugate comprising the effector moiety according to theinvention.

An embodiment within the third series of aspects and embodiments of theinvention is the antibody-drug conjugate or the ligand-drug conjugateaccording to the invention, wherein the antibody can bind to any one ofCD71, CA125, EpCAM(17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin,syndecan-1, vascular integrin alpha-V beta-3, HER2, EGFR, CD20, CD22,Folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, PSMA,CanAg, integrin-alphaV, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239,CD70, CD123, CD352, DLL3, CD25, ephrinA4, MUC1, Trop2, CEACAM5, CEACAM6,HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1,CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2, preferably CD71, HER2, EGFR, and/orwherein the antibody of the antibody-drug conjugate is or comprises anyone of cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab,brentuximab, inotuzumab, moxetumomab, polatuzumab, obinutuzumab, OKT-9anti-CD71 monoclonal antibody of the IgG type, pertuzumab, rituximab,ofatumumab, Herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38monoclonal antibody, an antibody of Table A2 or Table A3 or Table A4,preferably cetuximab or trastuzumab or OKT-9, or at least one tumor-cellspecific receptor binding-fragment thereof and/or at least onetumor-cell specific receptor binding-domain thereof, and/or wherein theantibody-drug conjugate comprises any one of Gemtuzumab ozogamicin,Brentuximab vedotin, Trastuzumab emtansine, Inotuzumab ozogamicin,Moxetumomab pasudotox and Polatuzumab vedotin and an antibody-drugconjugate of Table A2 and Table A3, and/or wherein the ligand-drugconjugate comprises or consists of at least one non-proteinaceous ligandand/or at least one proteinaceous ligand for binding to a cell-surfacemolecule such as EGF or a cytokine.

An aspect of the invention within the third series of aspects andembodiments of the invention relates to a therapeutic combinationcomprising: (a) the effector moiety according to the invention andoptionally a pharmaceutically acceptable excipient; and (b) anantibody-drug conjugate or a ligand-drug conjugate, and optionally apharmaceutically acceptable excipient.

An embodiment within the third series of aspects and embodiments of theinvention is the therapeutic combination according to the invention,wherein the antibody-drug conjugate can bind to any one of tumor-cellreceptors CD71, CA125, EpCAM(17-1A), CD52, CEA, CD44v6, FAP, EGF-IR,integrin, syndecan-1, vascular integrin alpha-V beta-3, HER2, EGFR,CD20, CD22, Folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor,PSMA, CanAg, integrin-alphaV, CA6, CD33, mesothelin, Cripto, CD3, CD30,CD239, CD70, CD123, CD352, DLL3, CD25, ephrinA4, MUC1, Trop2, CEACAM5,CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7,PD-L1, CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2, preferably CD71, HER2, EGFR,and/or wherein the antibody of the antibody-drug conjugate is orcomprises any one of cetuximab, daratumumab, gemtuzumab, trastuzumab,panitumumab, brentuximab, inotuzumab, moxetumomab, polatuzumab,obinutuzumab, OKT-9 anti-CD71 monoclonal antibody of the IgG type,pertuzumab, rituximab, ofatumumab, Herceptin, alemtuzumab, pinatuzumab,OKT-10 anti-CD38 monoclonal antibody, an antibody of Table A2 or TableA3 or Table A4, preferably cetuximab or trastuzumab or OKT-9, or atleast one tumor-cell specific receptor binding-fragment thereof and/orat least one tumor-cell specific receptor binding-domain thereof, and/orwherein the antibody-drug conjugate comprises any one of Gemtuzumabozogamicin, Brentuximab vedotin, Trastuzumab emtansine, Inotuzumabozogamicin, Moxetumomab pasudotox and Polatuzumab vedotin and anantibody-drug conjugate of Table A2 and Table A3, and/or wherein theligand-drug conjugate comprises or consists of at least onenon-proteinaceous ligand and/or at least one proteinaceous ligand forbinding to a cell-surface molecule such as EGF or a cytokine.

An aspect of the invention within the third series of aspects andembodiments of the invention relates to a pharmaceutical compositioncomprising the effector moiety according to the invention or theantibody-drug conjugate according to the invention or the ligand-drugconjugate according to the invention, and optionally a pharmaceuticallyacceptable excipient.

An aspect of the invention within the third series of aspects andembodiments of the invention relates to the effector moiety according tothe invention or the antibody-drug conjugate according to the inventionor the therapeutic combination according to the invention or theligand-drug conjugate according to the invention or the pharmaceuticalcomposition according to the invention, for use as a medicament.

An aspect of the invention within the third series of aspects andembodiments of the invention relates to the effector moiety according tothe invention or the antibody-drug conjugate according to the inventionor the ligand-drug conjugate according to the invention or thetherapeutic combination according to the invention or the pharmaceuticalcomposition according to the invention, for use in the treatment orprevention of a cancer or an autoimmune disease.

An aspect of the invention within a fourth series of aspects andembodiments of the invention relates to a conjugate comprising acell-surface molecule targeting molecule and at least one effectormoiety and further comprising at least one covalently bound saponin.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the at least onesaponin is a triterpenoid saponin and/or a bisdesmosidic triterpenesaponin belonging to the type of a 12,13-dehydrooleanane with analdehyde function in position C-23 and optionally comprising aglucuronic acid function in a carbohydrate substituent at the C-3beta-OHgroup of the saponin, and/or a saponin isolated from any one or more ofa Gypsophila species and/or a Saponaria species and/or an Agrostemmaspecies and/or a Quillaja species such as Quillaja saponaria.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the at least onesaponin is a single specific saponin or is a mixture of two or moredifferent saponins.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the at least onesaponin has a molecular mass of 3.000 Dalton or less, preferably 2.500Dalton or less, more preferably 2.300 Dalton or less, most preferably,2.000 Dalton or less, such as 1.500 Dalton-1.900 Dalton.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the at least onesaponin is one or more of the saponins in Table A1 or Scheme I, SO1861,SA1657, GE1741, SA1641, QS-21, QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B,QS-21 B-api, QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl,QS1861, QS1862, Quillajasaponin, Saponinum album, QS-18, Quil-A, Gyp1,gypsoside A, AG1, AG2, SO1542, SO1584, SO1658, SO1674, SO1832, or any oftheir stereomers and/or any combinations thereof, preferably the saponinis SO1861 and/or GE1741 and/or SA1641 and/or QS-21 and/or saponin with aquillaic acid aglycon core, a Gal-(1→2)-[Xyl-(1→3)]-GlcA carbohydratesubstituent at the C-3beta-OH group and aGlc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuccarbohydrate substituent at the C-28-OH group, and/or is3-O-beta-D-galactopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)]-beta-D-glucuronopyranosylquillaic acid28-O-beta-D-glucopyranosyl-(1→3)-beta-D-xylopyranosyl-(1→4)-alpha-L-rhamnopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)-4-OAc-beta-D-quinovopyranosyl-(1→4)]-beta-D-fucopyranoside,more preferably the at least one saponin is SO1861 and/or QS-21.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the at least onesaponin is a bisdesmosidic saponin having a molecular mass of at least1.500 Dalton and comprising an oleanan-type triterpene containing analdehyde group at the C-23 position and optionally a hydroxyl group atthe C-16 position, with a first branched carbohydrate side chain at theC-3 position which first branched carbohydrate side chain optionallycontains glucuronic acid, wherein the saponin contains an ester groupwith a second branched carbohydrate side chain at the C-28 positionwhich second branched carbohydrate chain preferably comprises at leastfour carbohydrate units, optionally containing at least one acetylresidue such as two acetyl residues and/or optionally comprising deoxycarbohydrates and/or optionally comprising quinovose and/or optionallycomprising glucose and/or optionally comprising 4-methoxycinnamic acidand/or optionally comprising5-O-[5-O-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoicacid and/or optionally comprising5-O-[5-O-Rha-(1→2)-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoicacid bound to a carbohydrate via an ester bond, or wherein the at leastone saponin is QS-21 or any one or more of QS-21A, QS-21 A-api, QS-21A-xyl, QS-21B, QS-21 B-api, QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api,QS-17-xyl, QS-18, QS1861, protonated QS1861 (QS1862), Quil-A.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the cell-surfacemolecule targeting molecule comprises or consists of a ligand or aproteinaceous ligand or a proteinaceous binding molecule for binding tothe cell-surface molecule.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the cell-surfacemolecule targeting molecule comprises or consists of a non-proteinaceousligand and/or a proteinaceous ligand for binding to a cell-surfacemolecule such as EGF or a cytokine, and/or comprises or consists of animmunoglobulin, at least one binding domain of an immunoglobulin and/orat least one binding fragment of an immunoglobulin, such as an antibody,an IgG, a molecule comprising or consisting of a Vhh domain or Vhdomain, a Fab, an scFv, an Fv, a dAb, an F(ab)₂, Fcab fragment, whichcan bind to the cell-surface molecule.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the cell-surfacemolecule targeting molecule can bind to a tumor-cell surface molecule,preferably a tumor-cell receptor such as a tumor-cell specific receptor,more preferably a receptor selected from CD71, CA125, EpCAM(17-1A),CD52, CEA, CD44v6, FAP, EGF-IR, integrin, syndecan-1, vascular integrinalpha-V beta-3, HER2, EGFR, CD20, CD22, Folate receptor 1, CD146, CD56,CD19, CD138, CD27L receptor, PSMA, CanAg, integrin-alphaV, CA6, CD33,mesothelin, Cripto, CD3, CD30, CD239, CD70, CD123, CD352, DLL3, CD25,ephrinA4, MUC1, Trop2, CEACAM5, CEACAM6, HER3, CD74, PTK7, Notch3, FGF2,C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1, CTLA4, CD52, PDGFRA, VEGFR1,VEGFR2, preferably selected from CD71, HER2 and EGFR.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the tumor-cellreceptor is internalized by the tumor cell after binding to thecell-surface molecule targeting molecule of the invention and theconjugate of the invention, and wherein preferably binding of theconjugate to the tumor-cell receptor is followed by tumor-cellreceptor-mediated internalization, e.g. via endocytosis, of a complex ofthe conjugate and the tumor-cell receptor.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the cell-surfacemolecule targeting molecule is or comprises a monoclonal antibody or atleast one cell-surface molecule binding fragment or -domain thereof, andpreferably comprises or consists of any one of cetuximab, daratumumab,gemtuzumab, trastuzumab, panitumumab, brentuximab, inotuzumab,moxetumomab, polatuzumab, obinutuzumab, OKT-9 anti-CD71 monoclonalantibody of the IgG type, pertuzumab, rituximab, ofatumumab, Herceptin,alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonal antibody, anantibody of Table A2 or Table A3 or Table A4, preferably cetuximab ortrastuzumab or OKT-9, or at least one cell-surface molecule bindingfragment or -domain thereof.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the at least oneeffector moiety comprises or consists of any one or more of anoligonucleotide, a nucleic acid and a xeno nucleic acid, preferablyselected from any one or more of a vector, a gene, a cell suicideinducing transgene, deoxyribonucleic acid (DNA), ribonucleic acid (RNA),anti-sense oligonucleotide (ASO, AON), short interfering RNA (siRNA),microRNA (miRNA), DNA aptamer, RNA aptamer, mRNA, mini-circle DNA,peptide nucleic acid (PNA), phosphoramidate morpholino oligomer (PMO),locked nucleic acid (LNA), bridged nucleic acid (BNA),2′-deoxy-2′-fluoroarabino nucleic acid (FANA), 2′-O-methoxyethyl-RNA(MOE), 2′-0,4′-aminoethylene bridged nucleic acid, 3′-fluoro hexitolnucleic acid (FHNA), a plasmid, glycol nucleic acid (GNA) and threosenucleic acid (TNA), or a derivative thereof, more preferably a BNA, forexample a BNA for silencing HSP27 protein expression.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the at least oneeffector moiety comprises or consists of at least one proteinaceousmolecule, preferably selected from any one or more of a peptide, aprotein, an enzyme such as urease and Cre-recombinase, aribosome-inactivating protein, a proteinaceous toxin selected from TableA5 and more preferably selected from any one or more of a viral toxinsuch as apoptin; a bacterial toxin such as Shiga toxin, Shiga-liketoxin, Pseudomonas aeruginosa exotoxin (PE) or exotoxin A of PE,full-length or truncated diphtheria toxin (DT), cholera toxin; a fungaltoxin such as alpha-sarcin; a plant toxin includingribosome-inactivating proteins and the A chain of type 2ribosome-inactivating proteins such as dianthin e.g. dianthin-30 ordianthin-32, saporin e.g. saporin-S3 or saporin-S6, bouganin orde-immunized derivative debouganin of bouganin, shiga-like toxin A,pokeweed antiviral protein, ricin, ricin A chain, modeccin, modeccin Achain, abrin, abrin A chain, volkensin, volkensin A chain, viscumin,viscumin A chain; or an animal or human toxin such as frog RNase, orgranzyme B or angiogenin from humans, or any fragment or derivativethereof; preferably the protein toxin is dianthin and/or saporin.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the at least oneeffector moiety comprises or consists of at least one payload,preferably selected from any one or more of a toxin targeting ribosomes,a toxin targeting elongation factors, a toxin targeting tubulin, a toxintargeting DNA and a toxin targeting RNA, more preferably any one or moreof emtansine, pasudotox, maytansinoid derivative DM1, maytansinoidderivative DM4, monomethyl auristatin E (MMAE, vedotin), monomethylauristatin F (MMAF, mafodotin), a Calicheamicin,N-Acetyl-γ-calicheamicin, a pyrrolobenzodiazepine (PBD) dimer, abenzodiazepine, a CC-1065 analogue, a duocarmycin, Doxorubicin,paclitaxel, docetaxel, cisplatin, cyclophosphamide, etoposide,docetaxel, 5-fluorouracyl (5-FU), mitoxantrone, a tubulysin, anindolinobenzodiazepine, AZ13599185, a cryptophycin, rhizoxin,methotrexate, an anthracycline, a camptothecin analogue, SN-38,DX-8951f, exatecan mesylate, truncated form of Pseudomonas aeruginosaexotoxin (PE38), a Duocarmycin derivative, an amanitin, a-amanitin, aspliceostatin, a thailanstatin, ozogamicin, tesirine, Amberstatin269 andsoravtansine, or a derivative thereof.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the at least oneeffector moiety is covalently bound to the cell-surface moleculetargeting molecule, either via at least one linker or bound directly tothe cell-surface molecule targeting molecule.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the at least oneeffector moiety is covalently bound to the cell-surface moleculetargeting molecule, thereby forming any one of antibody-drug conjugatesGemtuzumab ozogamicin, Brentuximab vedotin, Trastuzumab emtansine,Inotuzumab ozogamicin, Moxetumomab pasudotox and Polatuzumab vedotin andan antibody-drug conjugate of Table A2 and Table A3.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the at least onesaponin is covalently bound to the cell-surface molecule targetingmolecule preferably an amino-acid residue of the cell-surface moleculetargeting molecule, via an aldehyde function in the saponin, and/or tothe at least one effector moiety preferably via an amino-acid residue inthe at least one effector moiety, via an aldehyde function in thesaponin, preferably an aldehyde function in position C-23 in abisdesmosidic triterpene saponin belonging to the type of a12,13-dehydrooleanane.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the aldehydefunction in the at least one saponin, preferably the aldehyde functionin position C-23 of the at least one saponin, is covalently coupled tolinker N-ε-maleimidocaproic acid hydrazide, which linker is covalentlycoupled via a thio-ether bond to a sulfhydryl group in the cell-surfacemolecule targeting molecule and/or in the at least one effector moiety,such as a sulfhydryl group of a cysteine.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the at least onesaponin is a bisdesmosidic triterpene saponin belonging to the type of a12,13-dehydrooleanane, with an aldehyde function in position C-23 andcomprising a glucuronic acid function in a carbohydrate substituent atthe C-3beta-OH group of the saponin, wherein the saponin is covalentlybound to an amino-acid residue of the cell-surface molecule targetingmolecule and/or to the at least one effector moiety via said glucuronicacid function and preferably via an amino-acid residue in the at leastone effector moiety.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the glucuronic acidfunction in the carbohydrate substituent at the C-3beta-OH group of theat least one saponin is covalently coupled to linker1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate, which linker is covalently coupled via anamide bond to an amine group in the cell-surface molecule targetingmolecule and/or in the at least one effector moiety, such as an aminegroup of a lysine or an N-terminus of the cell-surface moleculetargeting molecule and/or of the at least one effector moiety.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the at least onesaponin is covalently bound to the cell-surface molecule targetingmolecule and/or to the at least one effector moiety either directly orvia at least one linker such as a bi-functional linker, for examplebased on N-ε-maleimidocaproic acid hydrazide and/or based on1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate, or a tri-functional linker, such as thetri-functional linker of Scheme II and Structure B.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the tri-functionallinker comprises a second chemical group with at least one saponincovalently bound thereto, a third chemical group for covalent binding tothe cell-surface molecule targeting molecule and a first chemical groupfor covalent binding to the at least one effector moiety, preferably thetri-functional linker is the trifunctional linker of Scheme II andStructure B.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the at least onesaponin is covalently bound to the cell-surface molecule targetingmolecule and to the at least one effector moiety via at least one linkercomprising a tri-functional linker to which tri-functional linker boththe cell-surface molecule targeting molecule and the at least oneeffector moiety are bound, preferably the tri-functional linker is thetrifunctional linker of Scheme II and Structure B.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the at least onelinker comprises at least one cleavable linker, wherein optionally saidcleavable linker is subject to cleavage under acidic, reductive,enzymatic or light-induced conditions, and preferably the cleavablelinker comprises a cleavable bond selected from a hydrazone bond or ahydrazide bond subject to cleavage under acidic conditions, and/or abond susceptible to proteolysis, for example proteolysis by Cathepsin B,and/or a bond susceptible for cleavage under reductive conditions suchas a disulphide bond.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the at least onelinker comprises at least one cleavable linker, wherein said cleavablelinker is subject to cleavage in vivo under acidic conditions as presentin endosomes and/or in lysosomes of mammalian cells, preferably of humancells, preferably at pH 4.0-6.5, and more preferably at pH≤5.5.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the at least onesaponin is covalently bound to a lysine side chain, forming an amidebond, and/or to a cysteine side chain, forming a thio-ether linkage or adisulphide bond, wherein the lysine and/or cysteine is/are comprised bythe cell-surface molecule targeting molecule and/or is/are comprised bythe at least one effector moiety, and wherein the at least one saponinis either directly bound to the lysine and/or cysteine, or is bound viaat least one linker optionally comprising a cleavable linker and/or atri-functional linker such as the tri-functional linker of Scheme II andStructure B.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the linker is basedon N-ε-maleimidocaproic acid hydrazide and/or based on1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate, a tri-functional linker such as thetri-functional linker of Scheme II and Structure B, a cleavable linker,and/or involves any one or more of a disulphide bond, a thio-ether bond,an amide bond, a hydrazide bond.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the at least onesaponin is covalently bound to the cell-surface molecule targetingmolecule and/or to the at least one effector moiety via at least onelinker, wherein the linker is or comprises a scaffold comprising apolymeric or oligomeric structure and further comprising at least onefourth chemical group for covalently coupling of the scaffold to thecell-surface molecule targeting molecule and/or to the at least oneeffector moiety.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the at least onesaponin is covalently bound to the polymeric or oligomeric structure ofthe scaffold via a cleavable bond and/or via a non-cleavable bond.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the cleavable bondis subject to cleavage under any of acidic conditions, reductiveconditions, enzymatic conditions and light-induced conditions, andpreferably the cleavable bond comprises a hydrazone bond or a hydrazidebond subject to cleavage under acidic conditions, and/or a bondsusceptible to proteolysis, for example proteolysis by Cathepsin B,and/or a bond susceptible for cleavage under reductive conditions suchas a disulphide bond.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the cleavable bondis subject to cleavage in vivo under acidic conditions as present inendosomes and/or in lysosomes of mammalian cells, preferably of humancells, preferably at pH 4.0-6.5, and more preferably at pH≤5.5.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the at least onesaponin is covalently bound to the polymeric or oligomeric structure ofthe scaffold via any one or more of an imine bond, a hydrazone bond, ahydrazide bond, an oxime bond, a 1,3-dioxolane bond, a disulphide bond,a thio-ether bond, an amide bond, a peptide bond or an ester bond,preferably via at least one linker.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the at least onesaponin is covalently bound to the polymeric or oligomeric structure ofthe scaffold via any one or more of an imine bond, a hydrazone bond anda hydrazide bond, which bond is preferably cleavable according to theinvention, wherein preferably the at least one saponin is covalentlybound to the polymeric or oligomeric structure of the scaffold via thealdehyde function in position C-23 of the at least one saponin.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the aldehydefunction in position C-23 of the at least one saponin is covalentlycoupled to linker N-ε-maleimidocaproic acid hydrazide, which linker iscovalently coupled via a thio-ether bond to a sulfhydryl group in thepolymeric or oligomeric structure of the scaffold, such as a sulfhydrylgroup of a cysteine.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the at least onesaponin is covalently bound to the polymeric or oligomeric structure ofthe scaffold via an amide bond, wherein preferably the at least onesaponin is covalently bound to the polymeric or oligomeric structure ofthe scaffold via the glucuronic acid function in the carbohydratesubstituent at the C-3beta-OH group of the at least one saponin, whenpresent.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the glucuronic acidfunction in the carbohydrate substituent at the C-3beta-OH group of theat least one saponin is covalently coupled to linker1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate, which linker is covalently coupled via anamide bond to an amine group in the polymeric or oligomeric structure ofthe scaffold, such as an amine group of a lysine or an N-terminus of thepolymeric or oligomeric structure of the scaffold.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the at least onesaponin is covalently bound to the polymeric or oligomeric structure ofthe scaffold, involving in the covalent bond the aldehyde function inposition C-23 of the at least one saponin, when present, and/orinvolving in the covalent bond the glucuronic acid function in thecarbohydrate substituent at the C-3beta-OH group of the at least onesaponin, when present.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the at least onefourth chemical group of the scaffold, for covalently coupling of thescaffold to the cell-surface molecule targeting molecule and/or to theat least one effector moiety, is a click chemistry group, preferablyselected from any one or more of a tetrazine, an azide, an alkene or analkyne, or a cyclic derivative of these groups, preferably an azidegroup.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the polymeric oroligomeric structure of the scaffold comprises a linear, branched and/orcyclic polymer, oligomer, dendrimer, dendron, dendronized polymer,dendronized oligomer, a DNA, a polypeptide, poly-lysine, a poly-ethyleneglycol, or an assembly of these polymeric or oligomeric structures whichassembly is preferably built up by covalent cross-linking.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the at least onesaponin is a defined number of saponins or a defined range of saponins,preferably 1-128 saponins or at least 2, 3, 4, 5, 6, 8, 10, 16, 32, 64or 128 saponins, or any number of saponins therein between, such as 7,9, 12 saponins.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the conjugatecomprises more than one saponin, preferably 2, 3, 4, 5, 6, 8, 10, 16,32, 64 or 1-100 saponins, or any number of saponins therein between,such as 7, 9, 12 saponins, covalently bound directly to an amino-acidresidue of the cell-surface molecule targeting molecule and/or to the atleast one effector moiety and preferably via an amino-acid residue inthe at least one effector moiety, preferably to a cysteine and/or to alysine, and/or covalently bound via at least one linker and/or via atleast one cleavable linker and/or via at least one polymeric oroligomeric scaffold of the invention, preferably 1-8 of such scaffoldsor 2-4 of such scaffolds, wherein 1-32 saponins, preferably 2, 3, 4, 5,6, 8, 10, 16 or 32 saponins, or any number of saponins therein between,such as 7, 9, 12 saponins, are covalently bound to the at least onescaffold.

An embodiment within the fourth series of aspects and embodiments of theinvention is the conjugate of the invention, wherein the at least onesaponin is covalently bound to the cell-surface molecule targetingmolecule and to the at least one effector moiety via a tri-functionallinker, the tri-functional linker comprising a second chemical groupwith at least one saponin covalently bound thereto either directly orvia a linker such as a cleavable linker and/or via the scaffoldcomprising a polymeric or oligomeric structure and a fourth chemicalgroup according to the invention for covalently coupling of the scaffoldto the tri-functional linker, the tri-functional linker furthercomprising a third chemical group for covalent binding to thecell-surface molecule targeting molecule and comprising a first chemicalgroup for covalent binding to the at least one effector moiety, whereinthe cell-surface molecule targeting molecule is bound to the thirdchemical group and/or the at least one effector moiety is bound to thefirst chemical group, preferably the trifunctional linker is thetrifunctional linker of Scheme II and Structure B.

An aspect of the invention within the fourth series of aspects andembodiments of the invention relates to a pharmaceutical compositioncomprising the conjugate of the invention and optionally apharmaceutically acceptable excipient and/or a pharmaceuticallyacceptable diluent.

An aspect of the invention within the fourth series of aspects andembodiments of the invention relates to the conjugate of the inventionor the pharmaceutical composition of the invention, for use as amedicament.

An aspect of the invention within the fourth series of aspects andembodiments of the invention relates to the conjugate of the inventionor the pharmaceutical composition of the invention, for use in thetreatment or prevention of a cancer or an autoimmune disease.

An aspect of the invention within a fifth series of aspects andembodiments of the invention relates to a therapeutic combination foruse as a medicament, wherein the therapeutic combination comprises: (a)a fourth pharmaceutical composition comprising a fourth proteinaceousmolecule comprising a binding site for binding to an epitope on acell-surface molecule and at least one saponin covalently bound to saidfourth proteinaceous molecule preferably to an amino-acid residue ofsaid fourth proteinaceous molecule, the fourth pharmaceuticalcomposition optionally further comprising a pharmaceutically acceptableexcipient; and (b) a fifth pharmaceutical composition comprising a fifthproteinaceous molecule, the fifth proteinaceous molecule comprising abinding site for binding to the epitope on the cell-surface molecule of(a) and an effector moiety, the fifth pharmaceutical compositionoptionally further comprising a pharmaceutically acceptable excipient,wherein the binding site of the fourth proteinaceous molecule and thebinding site of the fifth proteinaceous molecule are the same, andwherein the cell-surface molecule and the epitope on the cell-surfacemolecule, to which the fourth proteinaceous molecule can bind, and thecell-surface molecule and the epitope on the cell-surface molecule, towhich the fifth proteinaceous molecule can bind, are the same.

An aspect of the invention within the fifth series of aspects andembodiments of the invention relates to the fourth pharmaceuticalcomposition of the invention for use as a medicament.

An aspect of the invention within the fifth series of aspects andembodiments of the invention relates to a therapeutic combination foruse in the treatment or prevention of a cancer in a human subject,wherein the therapeutic combination comprises: (a) the fourthpharmaceutical composition of the invention; and (b) the fifthpharmaceutical composition of the invention, wherein the cell-surfacemolecule is expressed on a tumor cell surface, and preferably thecell-surface molecule is a tumor cell-specific surface molecule, andwherein preferably the epitope is a tumor-cell specific epitope.

An aspect of the invention within the fifth series of aspects andembodiments of the invention relates to the fourth pharmaceuticalcomposition of the invention, for use in the treatment or prophylaxis ofa cancer in a patient in need thereof, wherein the cell-surface moleculeis expressed on a tumor cell surface, and preferably the cell-surfacemolecule is a tumor cell-specific surface molecule, and whereinpreferably the epitope is a tumor-cell specific epitope.

An embodiment within the fifth series of aspects and embodiments of theinvention is the fourth pharmaceutical composition for use according tothe invention or the therapeutic combination for use of the invention,wherein the fifth pharmaceutical composition of the invention and thefourth pharmaceutical composition are administered to the patient inneed thereof.

An embodiment within the fifth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention, orthe fourth pharmaceutical composition for use according to theinvention, wherein the binding site of the fourth proteinaceous moleculeand the fifth proteinaceous molecule comprises or consists of animmunoglobulin or a binding fragment or binding domain of saidimmunoglobulin, such as any one or more of an antibody, an IgG, amolecule comprising or consisting of a Vhh domain or Vh domain, a Fab,an scFv, an Fv, a dAb, an F(ab)2, Fcab fragment, and/or comprises orconsists of at least one ligand, the ligand for binding to acell-surface molecule such as EGF or a cytokine.

An embodiment within the fifth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention, orthe fourth pharmaceutical composition for use according to theinvention, wherein the tumor-cell surface molecule is a cell-surfacereceptor specifically present at a tumor cell, and wherein preferablythe epitope is a tumor-cell specific epitope.

An embodiment within the fifth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention, orthe fourth pharmaceutical composition for use according to theinvention, wherein the at least one saponin is a triterpenoid saponinand/or a bisdesmosidic triterpene saponin belonging to the type of a12,13-dehydrooleanane with an aldehyde function in position C-23 andoptionally comprising a glucuronic acid function in a carbohydratesubstituent at the C-3beta-OH group of the saponin, and/or a saponinisolated from a Gypsophila species and/or a Saponaria species and/or anAgrostemma species and/or a Quillaja species such as Quillaja saponaria.

An embodiment within the fifth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention, orthe fourth pharmaceutical composition for use according to theinvention, wherein the at least one saponin is a single specific saponinor is a mixture of two or more different saponins, such as one or moreof the saponins in Table A1 or Scheme I, SO1861, SA1657, GE1741, SA1641,QS-21, QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B, QS-21 B-api, QS-21B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862,Quillajasaponin, Saponinum album, QS-18, Quil-A, Gyp1, gypsoside A, AG1,AG2, SO1542, SO1584, SO1658, SO1674, SO1832, or any of their stereomersand/or any combinations thereof, preferably the saponin is SO1861 and/orGE1741 and/or SA1641 and/or QS-21 and/or saponin with a quillaic acidaglycon core, a Gal-(1→2)-[Xyl-(1→3)]-GlcA carbohydrate substituent atthe C-3beta-OH group and aGlc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuccarbohydrate substituent at the C-28-OH group, and/or is3-O-beta-D-galactopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)]-beta-D-glucuronopyranosylquillaic acid28-O-beta-D-glucopyranosyl-(1→3)-beta-D-xylopyranosyl-(1→4)-alpha-L-rhamnopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)-4-OAc-beta-D-quinovopyranosyl-(1→4)]-beta-D-fucopyranoside,more preferably the saponin is SO1861 and/or QS-21.

An embodiment within the fifth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention, orthe fourth pharmaceutical composition for use according to theinvention, wherein the at least one saponin is a bisdesmosidic saponinhaving a molecular mass of at least 1.500 Dalton and comprising anoleanan-type triterpene containing an aldehyde group at the C-23position and optionally a hydroxyl group at the C-16 position, with afirst branched carbohydrate side chain at the C-3 position which firstbranched carbohydrate side chain optionally contains glucuronic acid,wherein the saponin contains an ester group with a second branchedcarbohydrate side chain at the C-28 position which second branchedcarbohydrate chain preferably comprises at least four carbohydrateunits, optionally containing at least one acetyl residue such as twoacetyl residues and/or optionally at least one deoxy carbohydrate and/ora quinovose and/or a glucose and/or 4-methoxycinnamic acid and/oroptionally comprising5-O-[5-O-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoicacid and/or optionally comprising5-O-[5-O-Rha-(1→2)-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoicacid bound to a carbohydrate via an ester bond, or wherein the at leastone saponin is QS-21 or any one or more of QS-21A, QS-21 A-api, QS-21A-xyl, QS-21B, QS-21 B-api, QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api,QS-17-xyl, QS-18, QS1861, protonated QS-1861 (QS1862), Quil-A.

An embodiment within the fifth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention, orthe fourth pharmaceutical composition for use according to theinvention, wherein the at least one saponin is a bisdesmosidictriterpene saponin belonging to the type of a 12,13-dehydrooleanane withan aldehyde function in position C-23, wherein the saponin is covalentlycoupled to the fourth proteinaceous molecule, preferably covalentlycoupled to an amino-acid residue of the fourth proteinaceous molecule,via an aldehyde function in the saponin, preferably said aldehydefunction in position C-23, preferably via at least one linker, and/orvia at least one cleavable linker, wherein the amino-acid residuepreferably is selected from cysteine and lysine.

An embodiment within the fifth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention, orthe fourth pharmaceutical composition for use according to theinvention, wherein the aldehyde function in position C-23 of the atleast one saponin is covalently coupled to linker N-ε-maleimidocaproicacid hydrazide, which linker is covalently coupled via a thio-ether bondto a sulfhydryl group in the fourth proteinaceous molecule, such as asulfhydryl group of a cysteine.

An embodiment within the fifth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention, orthe fourth pharmaceutical composition for use according to theinvention, wherein the at least one saponin is a bisdesmosidictriterpene saponin belonging to the type of a 12,13-dehydrooleanane withan aldehyde function in position C-23 and comprising a glucuronic acidfunction in a carbohydrate substituent at the C-3beta-OH group of thesaponin, wherein the saponin is covalently coupled to an amino-acidresidue of the fourth proteinaceous molecule via the glucuronic acidfunction in the carbohydrate substituent at the C-3beta-OH group of thesaponin, preferably via at least one linker, wherein the amino-acidresidue preferably is selected from cysteine and lysine.

An embodiment within the fifth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention, orthe fourth pharmaceutical composition for use according to theinvention, wherein the glucuronic acid function in the carbohydratesubstituent at the C-3beta-OH group of the at least one saponin iscovalently coupled to linker1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate, which linker is covalently coupled via anamide bond to an amine group in the fourth proteinaceous molecule, suchas an amine group of a lysine or an N-terminus of the fourthproteinaceous molecule.

An embodiment within the fifth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention, orthe fourth pharmaceutical composition for use according to theinvention, wherein the epitope is an epitope on a tumor-cell receptor,preferably a tumor-cell specific epitope, and wherein the receptor ispreferably a tumor-cell specific receptor, more preferably a receptorselected from CD71, CA125, EpCAM(17-1A), CD52, CEA, CD44v6, FAP, EGF-IR,integrin, syndecan-1, vascular integrin alpha-V beta-3, HER2, EGFR,CD20, CD22, Folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor,PSMA, CanAg, integrin-alphaV, CA6, CD33, mesothelin, Cripto, CD3, CD30,CD239, CD70, CD123, CD352, DLL3, CD25, ephrinA4, MUC1, Trop2, CEACAM5,CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7,PD-L1, CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2, most preferably selectedfrom CD71, HER2 and EGFR.

An embodiment within the fifth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention, orthe fourth pharmaceutical composition for use according to theinvention, wherein the tumor-cell receptor, preferably the tumor-cellspecific receptor, is internalized by the tumor cell after binding tothe fourth proteinaceous molecule of the invention and/or after bindingto the fifth proteinaceous molecule of the invention, and whereinpreferably binding of the fourth proteinaceous molecule and/or the fifthproteinaceous molecule to the tumor-cell receptor, such as thetumor-cell specific receptor, is followed by tumor-cellreceptor-mediated internalization, e.g. via endocytosis, of a complex ofthe fourth proteinaceous molecule and the tumor-cell receptor and of acomplex of the fifth proteinaceous molecule and the tumor-cell receptor.

An embodiment within the fifth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention, orthe fourth pharmaceutical composition for use according to theinvention, wherein the binding site of the fourth proteinaceous moleculeand the fifth proteinaceous molecule comprises a monoclonal antibody orat least one of a cell-surface molecule binding domain and/or -fragmentthereof, and preferably comprise or consist of any one of cetuximab,daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab,inotuzumab, moxetumomab, polatuzumab, obinutuzumab, OKT-9 anti-CD71monoclonal antibody of the IgG type, pertuzumab, rituximab, ofatumumab,Herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonalantibody, an antibody of Table A2 or Table A3 or Table A4, preferablycetuximab or trastuzumab or OKT-9, or at least one cell-surface moleculebinding fragment and/or -domain thereof, with the proviso that thefourth binding site is the same as the fifth binding site.

An embodiment within the fifth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention,wherein the effector moiety comprised by the fifth proteinaceousmolecule comprises or consists of any one or more of an oligonucleotide,a nucleic acid and a xeno nucleic acid, preferably selected from any oneor more of a vector, a gene, a cell suicide inducing transgene,deoxyribonucleic acid (DNA), ribonucleic acid (RNA), anti-senseoligonucleotide (ASO, AON), short interfering RNA (siRNA), microRNA(miRNA), DNA aptamer, RNA aptamer, mRNA, mini-circle DNA, peptidenucleic acid (PNA), phosphoramidate morpholino oligomer (PMO), lockednucleic acid (LNA), bridged nucleic acid (BNA),2′-deoxy-2′-fluoroarabino nucleic acid (FANA), 2′-O-methoxyethyl-RNA(MOE), 2′-0,4′-aminoethylene bridged nucleic acid, 3′-fluoro hexitolnucleic acid (FHNA), a plasmid, glycol nucleic acid (GNA) and threosenucleic acid (TNA), or a derivative thereof, more preferably a BNA, forexample a BNA for silencing HSP27 protein expression.

An embodiment within the fifth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention,wherein the effector moiety comprised by the fifth proteinaceousmolecule comprises or consists of at least one proteinaceous molecule,preferably selected from any one or more of a peptide, a protein, anenzyme such as urease and Cre-recombinase, a proteinaceous toxin, aribosome-inactivating protein, a protein toxin selected from Table A5and/or a bacterial toxin, a plant toxin, more preferably selected fromany one or more of a viral toxin such as apoptin; a bacterial toxin suchas Shiga toxin, Shiga-like toxin, Pseudomonas aeruginosa exotoxin (PE)or exotoxin A of PE, full-length or truncated diphtheria toxin (DT),cholera toxin; a fungal toxin such as alpha-sarcin; a plant toxinincluding ribosome-inactivating proteins and the A chain of type 2ribosome-inactivating proteins such as dianthin e.g. dianthin-30 ordianthin-32, saporin e.g. saporin-S3 or saporin-S6, bouganin orde-immunized derivative debouganin of bouganin, shiga-like toxin A,pokeweed antiviral protein, ricin, ricin A chain, modeccin, modeccin Achain, abrin, abrin A chain, volkensin, volkensin A chain, viscumin,viscumin A chain; or an animal or human toxin such as frog RNase, orgranzyme B or angiogenin from humans, or any fragment or derivativethereof; preferably the protein toxin is dianthin and/or saporin.

An embodiment within the fifth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention,wherein the effector moiety comprised by the fifth proteinaceousmolecule comprises or consists of at least one payload, preferablyselected from any one or more of a toxin targeting ribosomes, a toxintargeting elongation factors, a toxin targeting tubulin, a toxintargeting DNA and a toxin targeting RNA, more preferably any one or moreof emtansine, pasudotox, maytansinoid derivative DM1, maytansinoidderivative DM4, monomethyl auristatin E (MMAE, vedotin), monomethylauristatin F (MMAF, mafodotin), a Calicheamicin,N-Acetyl-y-calicheamicin, a pyrrolobenzodiazepine (PBD) dimer, abenzodiazepine, a CC-1065 analogue, a duocarmycin, Doxorubicin,paclitaxel, docetaxel, cisplatin, cyclophosphamide, etoposide,docetaxel, 5-fluorouracyl (5-FU), mitoxantrone, a tubulysin, anindolinobenzodiazepine, AZ13599185, a cryptophycin, rhizoxin,methotrexate, an anthracycline, a camptothecin analogue, SN-38,DX-8951f, exatecan mesylate, truncated form of Pseudomonas aeruginosaexotoxin (PE38), a Duocarmycin derivative, an amanitin, a-amanitin, aspliceostatin, a thailanstatin, ozogamicin, tesirine, Amberstatin269 andsoravtansine, or a derivative thereof.

An embodiment within the fifth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention,wherein the fifth proteinaceous molecule comprises or consists of anyone of Gemtuzumab ozogamicin, Brentuximab vedotin, Trastuzumabemtansine, Inotuzumab ozogamicin, Moxetumomab pasudotox and Polatuzumabvedotin and an antibody-drug conjugate of Table A2 and Table A3, or atleast one tumor-cell receptor binding-domain thereof and/or at least onetumor-cell receptor binding-fragment thereof, wherein said domain(s) orfragment(s) comprise(s) the effector moiety and are preferably (a)tumor-cell specific receptor binding-domain(s) and/or (a) tumor-cellspecific receptor binding-fragment(s).

An embodiment within the fifth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention,wherein the fourth proteinaceous molecule comprises more than onecovalently bound saponin, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32, 64,128 or 1-100 saponins, or any number of saponins therein between, suchas 7, 9, 12 saponins.

An embodiment within the fifth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention,wherein the at least one saponin is covalently bound directly to anamino-acid residue of the fourth proteinaceous molecule, preferably to acysteine and/or to a lysine, and/or is covalently bound via at least onelinker and/or via at least one cleavable linker and/or via at least oneoligomeric or polymeric scaffold, preferably 1-8 of such scaffolds or2-4 of such scaffolds, wherein the at least one scaffold is optionallybased on a dendron, wherein 1-32 saponins, such as 2, 3, 4, 5, 6, 8, 10,16, 32 saponins, or any number of saponins therein between, such as 7,9, 12 saponins, are covalently bound to the at least one scaffold.

An embodiment within the fifth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention,wherein the cleavable linker is subject to cleavage under acidicconditions, reductive conditions, enzymatic conditions or light-inducedconditions, and preferably the cleavable linker comprises a cleavablebond selected from a hydrazone bond and a hydrazide bond subject tocleavage under acidic conditions, and/or a bond susceptible toproteolysis, for example proteolysis by Cathepsin B, and/or a bondsusceptible for cleavage under reductive conditions such as a disulphidebond.

An embodiment within the fifth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention,wherein the cleavable linker is subject to cleavage in vivo under acidicconditions as present in endosomes and/or lysosomes of mammalian cells,preferably human cells, preferably at pH 4.0-6.5, and more preferably atpH≤5.5.

An embodiment within the fifth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention,wherein the oligomeric or polymeric scaffold comprises a polymeric oroligomeric structure and comprises at least one chemical group, the atleast one chemical group for covalently coupling of the scaffold to theamino-acid residue of said fourth proteinaceous molecule.

An embodiment within the fifth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention,wherein the at least one saponin is covalently bound to the polymeric oroligomeric structure of the scaffold via a cleavable linker according tothe invention.

An embodiment within the fifth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention,wherein the at least one saponin is covalently bound to the polymeric oroligomeric structure of the scaffold via any one or more of an iminebond, a hydrazone bond, a hydrazide bond, an oxime bond, a 1,3-dioxolanebond, a disulphide bond, a thio-ether bond, an amide bond, a peptidebond or an ester bond, preferably via at least one linker.

An embodiment within the fifth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention,wherein the at least one saponin comprises an aldehyde function inposition C-23 and optionally a glucuronic acid function in thecarbohydrate substituent at the C-3beta-OH group of the at least onesaponin, which aldehyde function is involved in the covalent bonding tothe polymeric or oligomeric structure of the scaffold, and/or, ifpresent, the glucuronic acid function is involved in the covalentbonding to the polymeric or oligomeric structure of the scaffold, eithervia direct binding or via at least one linker.

An embodiment within the fifth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention,wherein the aldehyde function in position C-23 of the at least onesaponin is covalently coupled to linker N-ε-maleimidocaproic acidhydrazide, which linker is covalently coupled via a thio-ether bond to asulfhydryl group in the polymeric or oligomeric structure of thescaffold, such as a sulfhydryl group of a cysteine.

An embodiment within the fifth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention,wherein the glucuronic acid function in the carbohydrate substituent atthe C-3beta-OH group of the at least one saponin is covalently coupledto linker1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate, which linker is covalently coupled via anamide bond to an amine group in the polymeric or oligomeric structure ofthe scaffold, such as an amine group of a lysine or an N-terminus of aproteinaceous molecule.

An embodiment within the fifth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention,wherein the at least one chemical group of the scaffold, for covalentlycoupling of the oligomeric or polymeric scaffold to the amino-acidresidue of said fourth proteinaceous molecule, is a click chemistrygroup, preferably selected from a tetrazine, an azide, an alkene or analkyne, or a cyclic derivative of these groups, more preferably theclick chemistry group is an azide.

An embodiment within the fifth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention,wherein the polymeric or oligomeric structure of the scaffold comprisesa linear, branched and/or cyclic polymer, oligomer, dendrimer, dendron,dendronized polymer, dendronized oligomer, a DNA, a polypeptide,poly-lysine, a poly-ethylene glycol, or an assembly of these polymericor oligomeric structures which assembly is preferably built up bycovalent cross-linking.

An embodiment within the fifth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention,wherein the fourth pharmaceutical composition and the fifthpharmaceutical composition are administered to the patient in needthereof.

An aspect of the invention within the fifth series of aspects andembodiments of the invention relates to the fourth pharmaceuticalcomposition of the invention, further comprising the fifth proteinaceousmolecule of the invention.

An aspect of the invention within the fifth series of aspects andembodiments of the invention relates to the fourth pharmaceuticalcomposition of the invention, further comprising the fifth proteinaceousmolecule of the invention, for use as a medicament.

An aspect of the invention within the fifth series of aspects andembodiments of the invention relates to the fourth pharmaceuticalcomposition of the invention, further comprising the fifth proteinaceousmolecule of the invention, for use in the treatment or prophylaxis ofcancer in a patient in need thereof.

An aspect of the invention within a sixth series of aspects andembodiments of the invention relates to a therapeutic combination foruse as a medicament, wherein the therapeutic combination comprises: (a)a sixth pharmaceutical composition comprising a sixth proteinaceousmolecule comprising a sixth binding site for binding to a sixthcell-surface molecule and at least one saponin covalently bound to saidsixth proteinaceous molecule preferably covalently bound to anamino-acid residue of said sixth proteinaceous molecule, the sixthpharmaceutical composition optionally further comprising apharmaceutically acceptable excipient; and (b) a seventh pharmaceuticalcomposition comprising a seventh proteinaceous molecule preferablydifferent from the sixth proteinaceous molecule, the seventhproteinaceous molecule comprising a seventh binding site for binding toa seventh cell-surface molecule different from the sixth cell-surfacemolecule and an effector moiety, the seventh pharmaceutical compositionoptionally further comprising a pharmaceutically acceptable excipient.

An aspect of the invention within the sixth series of aspects andembodiments of the invention relates to the sixth pharmaceuticalcomposition of the invention for use as a medicament.

An aspect of the invention within the sixth series of aspects andembodiments of the invention relates to a therapeutic combination foruse in the treatment or prevention of cancer in a human subject, whereinthe therapeutic combination comprises: (a) the sixth pharmaceuticalcomposition of the invention, wherein the sixth cell-surface molecule isa sixth tumor-cell surface molecule, preferably a sixth tumorcell-specific surface molecule; and (b) the seventh pharmaceuticalcomposition of the invention, wherein the seventh cell-surface moleculeis a seventh tumor-cell surface molecule different from the sixthtumor-cell surface molecule, preferably the seventh cell-surfacemolecule is a seventh tumor cell-specific surface molecule differentfrom the sixth tumor cell-specific surface molecule.

An aspect of the invention within the sixth series of aspects andembodiments of the invention relates to the sixth pharmaceuticalcomposition of the invention, for use in the treatment or prophylaxis ofcancer in a patient in need thereof, wherein the sixth cell-surfacemolecule is a sixth tumor-cell surface molecule, preferably a sixthtumor cell-specific surface molecule.

An embodiment within the sixth series of aspects and embodiments of theinvention is the sixth pharmaceutical composition for use according tothe invention or the therapeutic combination of the invention, whereinthe seventh pharmaceutical composition of the invention and the sixthpharmaceutical composition are administered to the patient in needthereof, and wherein the seventh tumor-cell surface molecule isdifferent from the sixth tumor-cell surface molecule, preferably theseventh tumor cell-specific surface molecule is different from the sixthtumor cell-specific surface molecule.

An embodiment within the sixth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention or thesixth pharmaceutical composition for use according to the invention,wherein the sixth binding site of the sixth proteinaceous moleculecomprises or consists of an immunoglobulin or at least one bindingfragment or -domain of said immunoglobulin for binding to the sixthcell-surface molecule, such as any one or more of an antibody, an IgG, amolecule comprising or consisting of a Vhh domain or Vh domain, a Fab,an scFv, an Fv, a dAb, an F(ab)2, Fcab fragment, and/or comprises orconsists of at least one ligand, preferably at least one ligand forbinding to the sixth cell-surface molecule such as EGF or a cytokine.

An embodiment within the sixth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention or thesixth pharmaceutical composition for use according to the invention,wherein the sixth binding site for binding to the sixth tumor-cellsurface molecule, preferably a tumor cell-specific surface molecule, isa sixth binding site for a sixth cell-surface receptor present at atumor cell, preferably specifically present at a tumor cell.

An embodiment within the sixth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention or thesixth pharmaceutical composition for use according to the invention,wherein the at least one saponin is a triterpenoid saponin or abisdesmosidic triterpene saponin, belonging to the type of a12,13-dehydrooleanane with an aldehyde function in position C-23 andoptionally comprising a glucuronic acid function in a carbohydratesubstituent at the C-3beta-OH group of the saponin, and/or a saponinisolated from a Gypsophila species and/or a Saponaria species and/or anAgrostemma species and/or a Quillaja species such as Quillaja saponaria.

An embodiment within the sixth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention or thesixth pharmaceutical composition for use according to the invention,wherein the at least one saponin is a single specific saponin or is amixture of two or more different saponins, such as one or more of thesaponins in Table A1 or Scheme I, SO1861, SA1657, GE1741, SA1641, QS-21,QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B, QS-21 B-api, QS-21 B-xyl,QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862,Quillajasaponin, Saponinum album, QS-18, Quil-A, Gyp1, gypsoside A, AG1,AG2, SO1542, SO1584, SO1658, SO1674, SO1832, or any of their stereomersand/or any combinations thereof, preferably the saponin is SO1861 and/orGE1741 and/or SA1641 and/or QS-21 and/or saponin with a quillaic acidaglycon core, a Gal-(1→2)-[Xyl-(1→3)]-GlcA carbohydrate substituent atthe C-3beta-OH group and aGlc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuccarbohydrate substituent at the C-28-OH group, and/or is3-O-beta-D-galactopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)]-beta-D-glucuronopyranosylquillaic acid28-O-beta-D-glucopyranosyl-(1→3)-beta-D-xylopyranosyl-(1→4)-alpha-L-rhamnopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)-4-OAc-beta-D-quinovopyranosyl-(1→4)]-beta-D-fucopyranoside,more preferably the saponin is SO1861 and/or QS-21.

An embodiment within the sixth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention or thesixth pharmaceutical composition for use according to the invention,wherein the at least one saponin is a bisdesmosidic saponin having amolecular mass of at least 1.500 Dalton and comprising an oleanan-typetriterpene containing an aldehyde group at the C-23 position andoptionally a hydroxyl group at the C-16 position, with a first branchedcarbohydrate side chain at the C-3 position which first branchedcarbohydrate side chain optionally contains glucuronic acid, wherein thesaponin contains an ester group with a second branched carbohydrate sidechain at the C-28 position which second branched carbohydrate chainpreferably comprises at least four carbohydrate units, optionallycontaining at least one acetyl residue such as two acetyl residuesand/or at least one deoxy carbohydrates and/or a quinovose and/or aglucose and/or 4-methoxycinnamic acid and/or optionally comprising5-O-[5-O-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoicacid and/or optionally comprising5-O-[5-O-Rha-(1→2)-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoicacid bound to a carbohydrate via an ester bond, or wherein the at leastone saponin is QS-21 or any one or more of QS-21A, QS-21 A-api, QS-21A-xyl, QS-21B, QS-21 B-api, QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api,QS-17-xyl, QS-18, QS1861, protonated QS1861 (QS1862), Quil-A.

An embodiment within the sixth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention or thesixth pharmaceutical composition for use according to the invention,wherein the at least one saponin is a bisdesmosidic triterpene saponinbelonging to the type of a 12,13-dehydrooleanane with an aldehydefunction in position C-23, wherein the saponin is covalently coupled thesixth proteinaceous molecule, preferably covalently coupled to anamino-acid residue of the sixth proteinaceous molecule, via an aldehydefunction in the saponin, preferably said aldehyde function in positionC-23, preferably via at least one linker, and/or via at least onecleavable linker, wherein the amino-acid residue preferably is selectedfrom cysteine and lysine.

An embodiment within the sixth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention or thesixth pharmaceutical composition for use according to the invention,wherein the aldehyde function in position C-23 of the at least onesaponin is covalently coupled to linker N-ε-maleimidocaproic acidhydrazide, which linker is covalently coupled via a thio-ether bond to asulfhydryl group in the sixth proteinaceous molecule, such as asulfhydryl group of a cysteine.

An embodiment within the sixth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention or thesixth pharmaceutical composition for use according to the invention,wherein the at least one saponin is a bisdesmosidic triterpene saponinbelonging to the type of a 12,13-dehydrooleanane with an aldehydefunction in position C-23 and comprising a glucuronic acid function in acarbohydrate substituent at the C-3beta-OH group of the saponin, whereinthe saponin is covalently coupled to an amino-acid residue of the sixthproteinaceous molecule via the glucuronic acid function in thecarbohydrate substituent at the C-3beta-OH group of the saponin,preferably via at least one linker, wherein the amino-acid residuepreferably is selected from cysteine and lysine.

An embodiment within the sixth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention or thesixth pharmaceutical composition for use according to the invention,wherein the glucuronic acid function in the carbohydrate substituent atthe C-3beta-OH group of the at least one saponin is covalently coupledto linker1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate, which linker is covalently coupled via anamide bond to an amine group in the sixth proteinaceous molecule, suchas an amine group of a lysine or an N-terminus of the sixthproteinaceous molecule.

An embodiment within the sixth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention or thesixth pharmaceutical composition for use according to the invention,wherein the seventh binding site of the seventh proteinaceous moleculecomprises or consists of an immunoglobulin, at least one binding domainof said immunoglobulin and/or at least one binding fragment of saidimmunoglobulin for binding to the seventh cell-surface molecule, such asan antibody, an IgG, a molecule comprising or consisting of a Vhh domainor Vh domain, a Fab, an scFv, an Fv, a dAb, an F(ab)2, Fcab fragment,and/or comprises or consists of at least one ligand, preferably a ligandfor binding to the seventh cell-surface molecule such as EGF or acytokine.

An embodiment within the sixth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention or thesixth pharmaceutical composition for use according to the invention,wherein the seventh binding site for binding to the seventh tumor-cellsurface molecule, preferably the seventh tumor cell-specific surfacemolecule, is a seventh binding site for a seventh cell-surface receptorpresent at a tumor cell, preferably specifically present at a tumorcell.

An embodiment within the sixth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention or thesixth pharmaceutical composition for use according to the invention,wherein the sixth binding site and the seventh binding site are bindingsites for binding to a sixth and seventh tumor-cell receptorrespectively, preferably for binding to a sixth and seventh tumor-cellspecific receptor respectively, preferably present at the same tumorcell, and wherein the sixth and seventh tumor-cell receptor arepreferably tumor-cell specific receptors, and/or are selected from CD71,CA125, EpCAM(17-1A), CD52, CEA, CD44v6, FAP, EGF-IR, integrin,syndecan-1, vascular integrin alpha-V beta-3, HER2, EGFR, CD20, CD22,Folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor, PSMA,CanAg, integrin-alphaV, CA6, CD33, mesothelin, Cripto, CD3, CD30, CD239,CD70, CD123, CD352, DLL3, CD25, ephrinA4, MUC1, Trop2, CEACAM5, CEACAM6,HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7, PD-L1,CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2, more preferably selected from HER2,CD71 and EGFR, with the proviso that the sixth binding site is differentfrom the seventh binding site and with the proviso that the sixth andseventh tumor-cell specific receptor, preferably the sixth and seventhtumor-cell specific receptor, are different receptors.

An embodiment within the sixth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention or thesixth pharmaceutical composition for use according to the invention,wherein the sixth binding site and the seventh binding site comprise orconsist of cetuximab, daratumumab, gemtuzumab, trastuzumab, panitumumab,brentuximab, inotuzumab, moxetumomab, polatuzumab, obinutuzumab, OKT-9anti-CD71 monoclonal antibody of the IgG type, pertuzumab, rituximab,ofatumumab, Herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38monoclonal antibody, an antibody of Table A2 or Table A3 or Table A4,preferably cetuximab or trastuzumab or OKT-9, or at least one tumor-cellreceptor binding-domain thereof and/or at least one tumor-cell receptorbinding-fragment thereof, with the proviso that the sixth binding siteis different from the seventh binding site.

An embodiment within the sixth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention or thesixth pharmaceutical composition for use according to the invention,wherein the sixth tumor-cell receptor is internalized by the tumor cellafter binding to the sixth proteinaceous molecule of the invention, andwherein preferably binding of the sixth proteinaceous molecule to thesixth tumor-cell receptor is followed by tumor-cell receptor-mediatedinternalization, e.g. via endocytosis, of a complex of the sixthproteinaceous molecule and the sixth tumor-cell receptor, wherein thesixth tumor-cell receptor is preferably a sixth tumor-cell specificreceptor.

An embodiment within the sixth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention,wherein the seventh tumor-cell receptor is internalized by the tumorcell after binding to the seventh proteinaceous molecule of theinvention, and wherein preferably binding of the seventh proteinaceousmolecule to the seventh tumor-cell receptor is followed by tumor-cellreceptor-mediated internalization, e.g. via endocytosis, of a complex ofthe seventh proteinaceous molecule and the seventh tumor-cell receptor,wherein the seventh tumor-cell receptor is preferably a seventhtumor-cell specific receptor.

An embodiment within the sixth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention or thesixth pharmaceutical composition for use according to the invention,wherein the effector moiety comprised by the seventh proteinaceousmolecule comprises or consists of at least one of an oligonucleotide, anucleic acid and a xeno nucleic acid, preferably selected from any oneor more of a vector, a gene, a cell suicide inducing transgene,deoxyribonucleic acid (DNA), ribonucleic acid (RNA), anti-senseoligonucleotide (ASO, AON), short interfering RNA (siRNA), microRNA(miRNA), DNA aptamer, RNA aptamer, mRNA, mini-circle DNA, peptidenucleic acid (PNA), phosphoramidate morpholino oligomer (PMO), lockednucleic acid (LNA), bridged nucleic acid (BNA),2′-deoxy-2′-fluoroarabino nucleic acid (FANA), 2′-O-methoxyethyl-RNA(MOE), 2′-0,4′-aminoethylene bridged nucleic acid, 3′-fluoro hexitolnucleic acid (FHNA), a plasmid, glycol nucleic acid (GNA) and threosenucleic acid (TNA), or a derivative thereof, more preferably a BNA, forexample a BNA for silencing HSP27 protein expression.

An embodiment within the sixth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention or thesixth pharmaceutical composition for use according to the invention,wherein the effector moiety comprised by the seventh proteinaceousmolecule comprises or consists of at least one proteinaceous molecule,preferably selected from any one or more of a peptide, a protein, anenzyme such as urease and Cre-recombinase, a proteinaceous toxin, aribosome-inactivating protein, a protein toxin selected from Table A5and/or a bacterial toxin, a plant toxin, more preferably selected fromany one or more of a viral toxin such as apoptin; a bacterial toxin suchas Shiga toxin, Shiga-like toxin, Pseudomonas aeruginosa exotoxin (PE)or exotoxin A of PE, full-length or truncated diphtheria toxin (DT),cholera toxin; a fungal toxin such as alpha-sarcin; a plant toxinincluding ribosome-inactivating proteins and the A chain of type 2ribosome-inactivating proteins such as dianthin e.g. dianthin-30 ordianthin-32, saporin e.g. saporin-S3 or saporin-S6, bouganin orde-immunized derivative debouganin of bouganin, shiga-like toxin A,pokeweed antiviral protein, ricin, ricin A chain, modeccin, modeccin Achain, abrin, abrin A chain, volkensin, volkensin A chain, viscumin,viscumin A chain; or an animal or human toxin such as frog RNase, orgranzyme B or angiogenin from humans, or any fragment or derivativethereof; preferably the protein toxin is dianthin and/or saporin.

An embodiment within the sixth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention or thesixth pharmaceutical composition for use according to the invention,wherein the effector moiety comprised by the seventh proteinaceousmolecule comprises or consists of at least one payload, preferablyselected from any one or more of a toxin targeting ribosomes, a toxintargeting elongation factors, a toxin targeting tubulin, a toxintargeting DNA and a toxin targeting RNA, more preferably any one or moreof emtansine, pasudotox, maytansinoid derivative DM1, maytansinoidderivative DM4, monomethyl auristatin E (MMAE, vedotin), monomethylauristatin F (MMAF, mafodotin), a Calicheamicin,N-Acetyl-γ-calicheamicin, a pyrrolobenzodiazepine (PBD) dimer, abenzodiazepine, a CC-1065 analogue, a duocarmycin, Doxorubicin,paclitaxel, docetaxel, cisplatin, cyclophosphamide, etoposide,docetaxel, 5-fluorouracyl (5-FU), mitoxantrone, a tubulysin, anindolinobenzodiazepine, AZ13599185, a cryptophycin, rhizoxin,methotrexate, an anthracycline, a camptothecin analogue, SN-38,DX-8951f, exatecan mesylate, truncated form of Pseudomonas aeruginosaexotoxin (PE38), a Duocarmycin derivative, an amanitin, a-amanitin, aspliceostatin, a thailanstatin, ozogamicin, tesirine, Amberstatin269 andsoravtansine, or a derivative thereof.

An embodiment within the sixth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention,wherein the seventh proteinaceous molecule comprises or consists of anyone of Gemtuzumab ozogamicin, Brentuximab vedotin, Trastuzumabemtansine, Inotuzumab ozogamicin, Moxetumomab pasudotox and Polatuzumabvedotin and an antibody-drug conjugate of Table A2 and Table A3, or atleast one tumor-cell receptor binding-domain thereof and/or at least onetumor-cell receptor binding-fragment thereof, wherein said domain(s) orfragment(s) comprise(s) the effector moiety and are preferably (a)tumor-cell specific receptor binding-domain(s) and/or (a) tumor-cellspecific receptor binding-fragment(s).

An embodiment within the sixth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention or thesixth proteinaceous molecule for use according to the invention, whereinthe sixth proteinaceous molecule comprises more than one covalentlybound saponin, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32, 64, 128 or 1-100saponins, or any number of saponins therein between, such as 7, 9, 12saponins.

An embodiment within the sixth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention or thesixth proteinaceous molecule for use according to the invention, whereinthe more than one covalently bound saponin are covalently bound directlyto an amino-acid residue of the sixth proteinaceous molecule, preferablyto a cysteine and/or to a lysine, and/or are covalently bound via atleast one linker and/or via at least one cleavable linker and/or via atleast one oligomeric or polymeric scaffold, preferably 1-8 of suchscaffolds or 2-4 of such scaffolds, wherein the at least one scaffold isoptionally based on a dendron, wherein 1-32 saponins, preferably 2, 3,4, 5, 6, 8, 10, 16, 32 saponins, or any number of saponins thereinbetween, such as 7, 9, 12 saponins, are covalently bound to the at leastone scaffold.

An embodiment within the sixth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention,wherein the cleavable linker is subject to cleavage under acidicconditions, reductive conditions, enzymatic conditions or light-inducedconditions, and preferably the cleavable linker comprises a cleavablebond selected from a hydrazone bond and a hydrazide bond subject tocleavage under acidic conditions, and/or a bond susceptible toproteolysis, for example proteolysis by Cathepsin B, and/or a bondsusceptible for cleavage under reductive conditions such as a disulphidebond.

An embodiment within the sixth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention,wherein the cleavable linker is subject to cleavage in vivo under acidicconditions as present in endosomes and/or lysosomes of mammalian cells,preferably human cells, preferably at pH 4.0-6.5, and more preferably atpH 5.5.

An embodiment within the sixth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention,wherein the oligomeric or polymeric scaffold comprises a polymeric oroligomeric structure and comprises at least one chemical group, the atleast one chemical group for covalently coupling of the scaffold to theamino-acid residue of said sixth proteinaceous molecule.

An embodiment within the sixth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention,wherein the at least one saponin is covalently bound to the polymeric oroligomeric structure of the scaffold via a cleavable linker according tothe invention.

An embodiment within the sixth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention,wherein the at least one saponin is covalently bound to the polymeric oroligomeric structure of the scaffold via any one or more of an iminebond, a hydrazone bond, a hydrazide bond, an oxime bond, a 1,3-dioxolanebond, a disulphide bond, a thio-ether bond, an amide bond, a peptidebond or an ester bond, preferably via at least one linker.

An embodiment within the sixth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention,wherein the at least one saponin comprises an aldehyde function inposition C-23 and optionally a glucuronic acid function in thecarbohydrate substituent at the C-3beta-OH group of the at least onesaponin, which aldehyde function is involved in the covalent bonding tothe polymeric or oligomeric structure of the scaffold, and/or, ifpresent, the glucuronic acid function is involved in the covalentbonding to the polymeric or oligomeric structure of the scaffold, eithervia direct binding or via at least one linker.

An embodiment within the sixth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention,wherein the aldehyde function in position C-23 of the at least onesaponin is covalently coupled to linker N-ε-maleimidocaproic acidhydrazide, which linker is covalently coupled via a thio-ether bond to asulfhydryl group in the polymeric or oligomeric structure of thescaffold, such as a sulfhydryl group of a cysteine.

An embodiment within the sixth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention,wherein the glucuronic acid function in the carbohydrate substituent atthe C-3beta-OH group of the at least one saponin is covalently coupledto linker1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxid hexafluorophosphate, which linker is covalently coupled via anamide bond to an amine group in the polymeric or oligomeric structure ofthe scaffold, such as an amine group of a lysine or an N-terminus of aproteinaceous molecule.

An embodiment within the sixth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention,wherein the chemical group of the polymeric or oligomeric scaffold, forcovalently coupling of the scaffold to the amino-acid residue of thesixth proteinaceous molecule, is a click chemistry group, preferablyselected from a tetrazine, an azide, an alkene or an alkyne, or a cyclicderivative of these groups, more preferably the click chemistry group isan azide.

An embodiment within the sixth series of aspects and embodiments of theinvention is the therapeutic combination for use of the invention,wherein the polymeric or oligomeric structure of the scaffold comprisesa linear, branched and/or cyclic polymer, oligomer, dendrimer, dendron,dendronized polymer, dendronized oligomer, a DNA, a polypeptide,poly-lysine, a poly-ethylene glycol, or an assembly of these polymericor oligomeric structures which assembly is preferably built up bycovalent cross-linking.

An aspect of the invention within the sixth series of aspects andembodiments of the invention relates to the therapeutic combination foruse of the invention, wherein the sixth pharmaceutical composition andthe seventh pharmaceutical composition are administered to the patientin need thereof.

An aspect of the invention within the sixth series of aspects andembodiments of the invention relates to the sixth pharmaceuticalcomposition of the invention, further comprising the seventhproteinaceous molecule of the invention.

An aspect of the invention within the sixth series of aspects andembodiments of the invention relates to the sixth pharmaceuticalcomposition of the invention, further comprising the seventhproteinaceous molecule of the invention, for use as a medicament.

An aspect of the invention within the sixth series of aspects andembodiments of the invention relates to the sixth pharmaceuticalcomposition of the invention, further comprising the seventhproteinaceous molecule of the invention, for use in the treatment orprophylaxis of cancer in a patient in need thereof.

Of course, any and all of a, b, c, d, e, f, g, h, I, j, k, m, n, p, q,r, s, t, u, v, w and/or x have the value in accordance with eachindividual embodiment and aspect of the invention for any and all of theaforementioned aspects and embodiments according to the invention. Inaddition, (tri-functional) linkers L1, L2, L4, L5, L6, L8, L9 and/orL10, if present in a molecule or conjugate or moiety of the invention,are the (tri-functional) linkers as indicated for each and any of theaforementioned aspects and embodiments of the invention, as is readilyappreciated by the skilled person. The oligomeric or polymeric scaffoldsL3 and/or L7, if present in a molecule or conjugate or moiety of theinvention, are the oligomeric or polymeric scaffolds as indicated foreach and any of the aforementioned aspects and embodiments of theinvention, as is also readily appreciated by the skilled person.Furthermore, the first ligand A1 and the first effector moiety B1, ifpresent, and the second ligand A2 and the second effector moiety B2, ifpresent, and the first effector moiety A1 and the first ligand B1, ifpresent, and the second effector moiety A2 and the second ligand B2, ifpresent, are the selected and indicated ligands and effector moieties,as disclosed for the first, second, third, fourth, fifth, and sixthseries of embodiment and aspects of the invention, and all furtherembodiments and aspects of the invention, outlined here above. Saponin Cis any one or more of the saponins referred to and listed in any of theaforementioned aspects and embodiments of the invention, in particularone or more saponins selected from Scheme I and/or Table A1.

An embodiment is the first, fourth or sixth proteinaceous molecule ofthe invention comprising a saponin comprising one or several or all ofthe indicated structural features of the saponin of Structure A inScheme I, the saponin of structure A referred to as a saponin with an‘ideal’ structure when endosomal escape enhancing activity towards aneffector moiety present in the endosome of a cell contacted with firstproteinaceous molecule, and/or a saponin selected from any one or moreof the further saponins in Scheme I:

According to the invention, a glycoside, such as a saponin C accordingto the invention, bound to the first, fourth and/or sixth proteinaceousmolecule of the invention, which has the ‘ideal’ structure for thepurpose of enhancing endosomal escape of an effector molecule bound tothe second or third or fifth or seventh proteinaceous molecule of theinvention is a bisdesmosidic saponin according to Structure A of SchemeI, having a molecular mass of at least 1.500 Dalton and comprising anoleanan-type triterpene containing an aldehyde group at the C-23position and optionally a hydroxyl group at the C-16 position, with afirst branched carbohydrate side chain at the C-3 position which firstbranched carbohydrate side chain optionally contains glucuronic acid,wherein the saponin contains an ester group with a second branchedcarbohydrate side chain at the C-28 position which second branchedcarbohydrate chain preferably comprises at least four carbohydrateunits, optionally containing at least one acetyl residue such as twoacetyl residues and/or optionally comprising deoxy carbohydrates and/oroptionally comprising quinovose and/or optionally comprising glucoseand/or optionally comprising 4-methoxycinnamic acid and/or optionallycomprising5-O-[5-O-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoicacid and/or optionally comprising5-O-[5-O-Rha-(1-2)-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoicacid bound to a carbohydrate via an ester bond.

SO1861 is different from the “ideal structure” displayed in Scheme I,Structure A, only in having only one acetyl residue at the quinovose andhaving an additional xylose. The “ideal structure” of a saponin forenhancing endosomal escape of an effector molecule or effector moiety ora payload, is a saponin which preferably has the Structure A of SchemeI, and saponins which display the endosomal escape enhancing activityhave one or more of the structural features displayed in Structure A ofScheme I. Without wishing to be bound by any theory, the inventorsbelief that the Structure A of Scheme I represents an “ideal saponin”(and not a minimum requirement saponin) for endosomal escape enhancingactivity, which means that not all of the structures (chemical groups)can or must be present in each saponin with at least sufficientendosomal escape enhancing activity to promote accumulation of theeffector moiety in the cytosol, and which means that some saponins mighthave other structure elements such as acyl chains, and/or for yet othersaponins that display endosomal escape enhancing activity, the sugarscan be different than the sugars displayed in Scheme I. For example, theQS-21 saponin and some of the saponins in the water soluble fraction ofQuillaja saponaria (Quillaja saponins; Quil-A) differ in thecarbohydrate modification at C-28 when the ideal structure of StructureA in Scheme I is considered: presence of an acyl chain in QS-21 forexample. In the water soluble fraction of Quillaja saponaria, saponinssuch as QS-7, QS1862, are similar to the ideal Structure A, and aresimilar to SO1861.

An embodiment is the first, fourth or sixth proteinaceous molecule ofthe invention, comprising the oligomeric tri-functional linker as thescaffold core structure, according to Scheme II:

wherein the saponins are covalently bound to the tri-functional linkerscaffold L9 and/or L10 via labile, cleavable hydrazone linkers (acidsensitive) and/or via a maleimide comprising bond, whereas the bindingof the scaffold to the binding site such as an antibody is establishedvia labile, cleavable hydrazone linkers (acid sensitive) and/or via amaleimide comprising bond with cysteines in the binding site such as 1,2, 3 or 4 cysteines, therewith forming Structure B:

such that 1-4 scaffolds are covalently bound to a single e.g. antibodysuch as a monoclonal antibody. According to the invention, one of thetwo displayed saponins in Structure B can also be replaced by acovalently coupled effector moiety, effector molecule, payload, or bothsaponins are absence with two covalently coupled effector moieties,effector molecules, payloads coupled to the tri-functional linker. Thebinding site is for example an antibody or a binding fragment or bindingdomain thereof.

The inventors established that the therapeutic window of an antibodydrug conjugate, such as the second and third and fifth and seventhproteinaceous molecules in the second or third or fifth or seventhpharmaceutical composition of the invention, respectively, increaseswhen administered to a tumor-bearing mammal (mouse) to whom also thefirst, fourth or sixth pharmaceutical composition is administered. Thefirst, fourth or sixth proteinaceous protein has at least one glycosidesuch as a saponin C bound thereto, preferably covalently, morepreferably via a cleavable linker. The saponin C augments thetherapeutic efficacy of the effector moiety A1, B1, A2 or B2, bound tothe second and third and fifth and seventh proteinaceous molecule,likely by enhancing the endosomal escape of the effector moiety into thecytosol where the activity of the effector moiety is desired. This way,already at a lower dose than the conventional dose of the ADC, i.e. thesecond or third or fifth or seventh proteinaceous molecule, therapeuticeffect is established under influence of the presence of the first,fourth or sixth proteinaceous molecule comprising the saponin C near, atand/or inside the targeted cell. The targeted cell is for example adiseased cell such as a tumor cell or an auto-immune cell or a B-celldisease related B-cell, etc. The effector moiety A1, B1, A2 or B2 is forexample a toxin as part of an ADC or an oligonucleotide such as a BNA aspart of an AOC according to the invention.

By targeting (two) different cell-surface molecules with the first andsecond, or fourth and fifth, or sixth and seventh proteinaceousmolecule, the delivery of the saponin C and the effector molecule A1,B1, A2 or B2 at and inside the cytosol of the very same targeted cell,exposing (both different) cell-surface molecules on the cell surface, isimproved and more specific, compared to exposure of such cells to onlythe second, third, fifth or seventh proteinaceous molecule such as anADC or an AOC, without the presence of the cell-targeted saponin C(first, fourth or sixth proteinaceous molecule). An aberrant cellselected for separate targeting by the binding site of the first, fourthor sixth proteinaceous molecule and by the binding site of the second,third, fifth or seventh proteinaceous molecule, wherein the bindingsites are different and wherein the epitope to which the first andsecond proteinacous molecules bind are different and are located in/on adifferent kind and type of cell-surface molecule such as two differentreceptors, ideally bears the first epitope and the second epitope on thefirst cell-surface molecule and the second cell-surface moleculerespectively, to a high extent (i.e. relatively higher expression of thetwo distinct and different cell-surface molecules on the targeted cellsuch as for example a tumor cell or an auto-immune cell, than theexpression on a non-targeted cell such as for example a healthy cell)and/or expose the first and second cell-surface molecules specifically,when (neighboring) healthy cells in a patient are considered.Preferably, both cell-surface molecules targeted by the first and secondbinding sites are relatively highly and/or specifically expressed on thetargeted (diseased, tumor) cell compared to healthy cells, which are notintended to be targeted with the molecules and conjugates of theinvention. An embodiment is the pharmaceutical combination, wherein atleast one of the first (fourth, sixth) and second (fifth, seventh)binding site and thus at least one of the first (fourth, sixth) andsecond (fifth, seventh) cell-surface molecule such as a first (fourth,sixth) and second (fifth, seventh) tumor-cell receptor, is expressedspecifically or to a relatively higher extent when compared toexpression of the first (fourth, sixth) cell-surface molecule and/or thesecond (fifth, seventh) cell-surface molecule on the surface of ahealthy (neighbouring) cell. Thus, the first (fourth, sixth) epitope orthe second (fifth, seventh) epitope, preferably the first (fourth,sixth) epitope and the second (fifth, seventh) epitope, on the targetedcell-surface molecule is/are ideally unique to the targeted diseasedcells, and is/are at least specifically present and exposed at thesurface of the targeted cells. Binding of the first (fourth, sixth) andsecond (fifth, seventh) proteinaceous molecules to their respectivefirst (fourth, sixth) and second (fifth, seventh) epitope on a targetedcell is followed by endocytosis of the complexes of the first (fourth,sixth) proteinaceous molecule and the first (fourth, sixth) targetcell-surface molecule and the second (fifth, seventh) proteinaceousmolecule and the second (fifth, seventh) target cell-surface molecule.Since the first and second proteinaceous molecules have to enter thesame target cell through binding interaction with two differentcell-surface molecules both expressed to a sufficient extent or uniquelyon the targeted cell when compared to healthy cells that should not betargeted, accumulation of a therapeutically active amount of first andsecond proteinaceous molecules inside the target cells is only possibleand occurring if expression levels of the two distinct targetedcell-surface molecules is both above a certain minimal expressionthreshold. At the same time, the fact that the effector moiety bound tothe second (fifth, seventh) proteinaceous molecule is only capable ofexerting its intracellular (e.g. cytotoxic or gene silencing) activityin the presence of the first (fourth, sixth) proteinaceous moleculebearing the covalently bound saponin, when both the first and secondproteinaceous molecules were capable to enter the target cell insufficient amounts by binding to sufficiently exposed and expressedfirst and second cell-surface molecules, also provides a safeguardagainst negative and undesired side effects of the effector moietytowards e.g. healthy cells and healthy tissue not meant to be targetedand affected by the effector moiety, when expression of at least on ofthe first and second cell-surface molecules is sufficiently low at thehealthy cells and preferably when expression of both the first andsecond targeted cell-surface molecules is sufficiently low at thehealthy cells. That is to say, sufficiently low expression or evenabsence of exposed first and second cell-surface molecules with regardto the first and second cell-surface molecules, and at least either thefirst cell-surface molecule or the second cell-surface molecule, boundby the first and second binding site of the first and secondproteinaceous molecules respectively, does ideally not allow entranceinto (non-targeted) healthy cells of both the first and secondproteinaceous molecules to amounts that would in concert result inendosomal escape of the effector moiety under influence of the saponinbound to the first proteinaceous molecule. Since the ADC or the AOC canbe used at lower dose compared to when the first proteinaceous moleculewas not added to the therapeutic regimen, ADC or AOC entrance in healthycells to low extent already bears a lower risk for occurrence ofunwanted side effects when for example the targeting and killing oftarget diseased cells such as tumor cells and auto-immune cells isconsidered.

Synchronization is the missing link between a successful deliverystrategy for mice and its application in humans. Indeed, the inventorsestablished in a series of in vivo mouse tumor models that separatelyadministering to the mice a dose of free saponin and a dose of ADC(second or third or fifth or seventh proteinaceous molecule according tothe invention) did not result in any desired anti-tumor activity such asdelayed tumor growth, tumor regression, diminished and slower tumorgrowth, compared to control animals not treated with the ADC and freesaponin. The free saponin was administered using various routes ofadministration and using various time points of administering the freesaponin compared to the moment of administering the ADC (administeringfree saponin before, during and after administering the ADC). The ADCtested in in vivo tumor models was cetuximab-dianthin (with freeSO1861), or trastuzumab-saporin (with free SO1861). Varying the dose offree saponin did not provide for an efficacious anti-tumor activity. TheADCs referred to were administered at a dose that in itself did notinflict any beneficial anti-tumor effect on the tumor-bearing animals.Surprisingly, the inventors now established that beneficial anti-tumoractivity in various in vitro mammalian cell-based bioassays and/or invarious in vivo animal tumor models can be achieved by treating theanimals with conjugates according to the invention, optionallycomprising a scaffold according to the invention, i.e. combinations offirst and second or first and third or fourth and fifth or sixth andseventh proteinaceous molecules of the invention. The scaffold forexample being a tri-functional linker with a covalently bound saponin(e.g. SO1861, QS-21) via a cleavable or non-cleavable linkage, and/orwith a covalently bound effector moiety (e.g. dianthin, silencing BNA(HSP27) via a non-cleavable bond or a cleavable bond, and/or with acovalently bound monoclonal antibody such as cetuximab, trastuzumab,OKT-9, or the scaffold being a dendron, such as a dendron to which forexample four moieties can bind such as four saponin molecules, or adendron for binding for example two saponins and two effector molecules,the dendron comprising a chemical group for (covalent) coupling to aligand or an antibody or fragment or domain thereof. Reference is madeto the Examples section, exemplifying various of these scaffoldsaccording to the invention, showing in vivo and/or in vitro anti-tumorcell activity when cell toxicity exerted by e.g. a proteinaceous toxinis considered or when gene silencing in the tumor cell is considered.

Without wishing to be bound by any theory, in view of the failuresobserved when treatment of tumor-bearing animals with an ADC togetherwith free saponin is considered, it is preferred to synchronize thepresence of both, the at least one saponin, and the effector moiety,preferably a toxin or an (antisense) oligonucleotide, in compartments orvesicles of the endocytic pathway of the target cell, e.g. a tumor cellor an auto-immune cell. With ADC (and/or AOC) and free saponin,synchronizing the presence of the molecules in the late endosomes, inorder to obtain the synergistic effects in vivo was not beneficiallyobtainable according to attempts of the inventors. In one aspect, theinvention preferably solves at least the following problem with respectto combining the effector moiety comprised by the second, third, fifthor seventh proteinaceous molecule and the saponins comprised by thefirst, fourth or sixth proteinaceous molecule: without wishing to bebound by any theory the only reasonable chemical group within, e.g., thesaponins that can be used for (covalent), in particular single andcleavable, retainable coupling is required for the endosomal escapeactivity. Known restrictions are most likely the reason why saponinshave not been used in combination with pharmaceutically activesubstances in clinical investigations other than the application ofsaponins in vaccination regimes wherein the use of animmune-potentiating adjuvant substance was implied, although thestriking endosomal escape enhancer effect of, e.g., saponins listed inTable A1 and Scheme I is known for more than 10 years. For exampleproviding a first, fourth or sixth proteinaceous molecule of theinvention with a covalently conjugated scaffold solves thesedifficulties, at least in part. Surprisingly, the saponins previouslyapplied for their immune-potentiating activity in the vaccinationcontext involving saponins as adjuvant component, are now also suitablyfor (covalent) coupling to the first, fourth or sixth proteinaceousmolecule of the invention, for anti-tumor activity in vitro and in vivo.

An effector moiety useful in the present invention preferably relies onlate endosomal escape for exerting its effect. Some effectors, such as,e.g., a pseudomonas exotoxin, are rerouted to other organelles prior tothe “late endosomal stage” and, thus, would normally not benefit fromcoupling to the second proteinaceous molecule according to the presentinvention. However, such toxin may be adapted for use with the presentinvention, e.g., by deleting the signal peptide responsible rerouting.In particular toxins that are highly toxic and would require only onemolecule to escape the endosomes to kill a cell maybe modified to beless potent. It is preferred to use a toxin that kills a cell if atleast 2, more preferably at least 5, more preferably at least 10, morepreferably at least 20, more preferably at least 50, most preferably atleast 100 toxin molecules escape the endosome. It is further preferredthat a second proteinaceous molecule of the invention comprises acovalently conjugated functionalized scaffold, i.e. a scaffoldcomprising covalently bound effector moietie(s) for targeting thescaffold comprising the bound effector moietie(s) at a target cell suchas a tumor cell or an auto-immune cellFurther, in order to reduceoff-target toxicity, cell membrane non-permeable small molecule toxinsare preferred effector molecules over cell membrane permeable toxins.

The term “ligand” as used in this invention has its ordinary meaning andpreferably means a molecule or structure that is able to bind anothermolecule or structure on the cell surface of a target cell, wherein saidmolecule or structure on the cell surface can be endocytosed and ispreferably absent or less prominent on off-target cells. Preferably,said molecule or structure on the cell surface is constitutivelyendocytosed. More preferably a ligand in this invention inducesendocytosis of said molecule or structure on the cell surface of targetcells after binding to said molecule or structure. This is for instancethe case for Epidermal Growth Factor Receptor (EGFR), present on thesurface of a variety of cancer cells. Examples of molecules orstructures on the cell surface of target cells that are constitutivelyendocytosed, are for instance Claudin-1 or major histocompatibilitycomplex class II glycoproteins. A ligand can, e.g., be an antibody, agrowth factor or a cytokine. Combining in a carrier molecule a toxinwith a ligand is one possibility to create a targeted toxin. A toxinthat is only toxic in a target cell because it interferes with processesthat occur in target cells only can also be seen as a targeted toxin (asin off-target cells it cannot exert its toxic action, e.g. apoptin).Preferably, a targeted toxin is a toxin that is combined with a ligandor e.g. a monoclonal antibody in order to be active in target cells andnot in off-target cells (as it is only bound to and endocytosed bytarget cells). In a functionalized scaffold comprising a carriermolecule comprising a ligand and an effector moiety (i.e. a second orthird proteinaceous molecule), the ligand or the monoclonal antibodyguides the effector moiety and scaffold to the target cells. Afterinternalization, the at least one glycoside, preferably a saponincomprised by the conjugate of the first proteinaceous molecule and thesaponin, mediates the endosomal escape of the effector moiety. Thesaponin is typically a saponin listed in Table A1 and Scheme I, andpreferably the saponin is SO1861 and/or QS-21, and/or SA1641 and/orGE1741.

The inventors established that immunoglobulins, domains thereof,ligands, etc., are particularly suitable for application as the (first)binding site of the first, fourth or sixth proteinaceous molecule (andthe same binding site of the third proteinaceous molecule) comprisingthe (first) binding site. Similarly, the inventors established thatimmunoglobulins, domains thereof, ligands, etc., are particularlysuitable for application as the second, fifth or seventh binding site ofthe second, fifth or seventh proteinaceous molecule comprising thesecond, fifth or seventh binding site. For example, antibodies andbinding domains of antibodies are suitable for targeting an epitope onthe exposed surface of a selected cell-surface molecule, resulting intargeting the first and third and fourth and sixth (and separately thesecond, fifth and seventh) proteinaceous molecule to target cellsexpressing the cell-surface molecule targeted by the first and third andfourth and sixth proteinaceous molecule and/or target also cellsexpressing the second or fifth or seventh cell-surface molecule targetedby the second or fifth or seventh proteinaceous molecule, these cellsalso expressing the first and third and fourth and sixth cell-surfacemolecule (which is the same cell-surface molecule), and having saidcell-surface molecules on their cell surface. Similarly, ligands such asEGF, targeting the EGFR on target cells, are suitable for application asthe binding site in the first and third and fourth and sixthproteinaceous molecules, or as the second or fifth or seventh bindingsite in the second or fifth or seventh proteinaceous molecule with theproviso that the second, fifth and seventh binding site is differentfrom the first and third and fourth and sixth binding site which firstand third binding site are the same. Preferred are binding sites for thefirst and third, fourth, sixth epitope or for the second, fifth, seventhepitope, which are specific for the binding of the first and third,fourth, sixth proteinaceous molecules to the first, fourth, sixthcell-surface molecule and/or for the binding of the second, fifth,seventh proteinaceous molecule to the second, fifth, seventhcell-surface molecule, the first (third, fourth, sixth) and second(fifth, seventh) cell-surface molecules exposed on the very same targetcell. Binding sites based on antibodies or domains or binding fragmentsthereof for example provide for such desired specificity for a selectedfirst, second, third, fourth, fifth, sixth, seventh epitope on aselected first or second or third (same as the first), fourth, fifth,sixth, seventh cell-surface molecule of a selected cell for targetingsuch as a diseased cell, a tumor cell, an auto-immune cell, etc.Therefore, first, second and third and fourth, fifth, sixth and seventhbinding sites based on antibodies or binding molecules (fragments,domains) are preferred for the first and second and third and fourth andfifth and sixth ad seventh proteinaceous molecules.

By targeting the same cell-surface molecule with the first and third,and fourth and fifth proteinaceous molecule, the delivery of the saponinC and the effector moiety A1, B1, A2 or B2 at and inside the cytosol ofthe very same targeted cell is improved and more specific. An aberrantcell selected for targeting by the binding site of the first and third,or the fourth and fifth proteinaceous molecule ideally bears thecell-surface molecule to a high extent and/or specifically, when(neighboring) healthy cells in a patient are considered. Thus, theepitope on the targeted cell-surface molecule is ideally unique to thetargeted diseases cells, and is at least specifically present andexposed at the surface of the targeted cells. Binding of the first andthird or fourth and fifth proteinaceous molecules is followed byendocytosis of the complexes of the first proteinaceous molecule and thetarget cell-surface molecule and the third proteinaceous molecule andthe target cell-surface molecule, or of the fourth proteinaceousmolecule and the target cell-surface molecule and the fifthproteinaceous molecule and the target cell-surface molecule. Since thefirst and third, or fourth and fifth, proteinaceous molecules have toenter the same target cell through binding interaction with the verysame cell-surface molecules, accumulation of a therapeutically activeamount of first and third, or fourth and fifth, proteinaceous moleculesinside the target cells is only possible and occurring if expressionlevels of the targeted cell-surface molecule is above a certain minimalexpression threshold. At the same time, the fact that the effectormoiety bound to the third and fifth proteinaceous molecule is onlycapable of exerting its intracellular (e.g. cytotoxic or gene silencing)activity in the presence of the first or fourth proteinaceous moleculebearing the covalently bound saponin, when both the first and third, orboth the fourth and fifth, proteinaceous molecules were capable to enterthe target cell in sufficient amounts by binding to sufficiently exposedand expressed cell-surface molecule, also provides a safeguard againstnegative and undesired side effects of the effector moiety towards e.g.healthy cells and healthy tissue not meant to be targeted and affectedby the effector moiety, when expression of the targeted cell-surfacemolecule is sufficiently low at the healthy cells. That is to say, lowexpression of the cell-surface molecule bound by the binding site of thefirst and third, or fourth and fifth, proteinaceous molecules, does notallow entrance of both the first and third, or both the fourth andfifth, proteinaceous molecules to amounts that would in concert resultin endosomal escape of the effector moiety under influence of thesaponin bound to the first and fourth proteinaceous molecule. Since theADC or AOC can be used at lower dose compared to when the first orfourth proteinaceous molecule was not added to the therapeutic regimen,ADC or AOC entrance in healthy cells to low extent already bears a lowerrisk for occurrence of unwanted side effects when for example thetargeting and killing of target diseased cells such as tumor cells andauto-immune cells is considered.

Throughout the description and claims (the whole application), the terms‘first’ and ‘third’ have the same meaning when the first and thirdepitope, the first and third binding site, the first and thirdcell-surface molecule are considered. That is to say, for the first andthird proteinaceous molecules, the targeted epitope is the same, thebinding site is the same, the targeted cell-surface molecule such as atumor-cell (specific) receptor is the same. The same for the terms‘fourth’ and ‘fifth’.

Tables A2, A3 and A4 list preferred examples of the first, third, fourthand sixth cell-surface molecule comprising the first (third, fourth andsixth) epitope for the first (third, fourth and sixth) binding site ofthe first (third, fourth and sixth) proteinaceous molecule. In addition,Tables A2, A3 and A4 also list preferred examples of the second, fifthand seventh cell-surface molecule comprising the second, fifth andseventh epitope for the second, fifth and seventh binding site of thesecond, fifth, seventh proteinaceous molecule. When the first, third,fourth, sixth and/or second, fifth, seventh cell-surface molecule isspecifically expressed on the target cell, preferably both the first,third, fourth and sixth, and the second, fifth, seventh cell-surfacemolecules, and when the first and second epitopes on the first andsecond cell-surface molecules respectively, to which the first bindingsite and/or the second binding site can bind respectively, isspecifically present in the first and/or second cell-surface molecule,specific targeting of the first, third and/or second proteinaceousmolecule to the same desired target cell such as a tumor cell exposingthe first and second tumor-cell surface molecules, is facilitated,whereas other cells such as healthy cells, which do not express thefirst and/or second cell-surface molecule or do express the first and/orsecond cell-surface molecule to a lower extent, preferably which do notexpress the first and second cell-surface molecule or do express thefirst and second cell-surface molecule to a lower extent compared toexpression of the cell-surface molecule(s) on the targeted (aberrant)cell, are not targeted by the first, third and second proteinaceousmolecule or are targeted to a lower extent.

A pharmaceutically active substance in this invention is an effectormoiety that is used to achieve a beneficial outcome in an organism,preferably a vertebrate, more preferably a human being such as a cancerpatient or an auto-immune patient. Benefit includes diagnosis,prognosis, treatment, cure and/or prevention of diseases and/orsymptoms. The pharmaceutically active substance may also lead toundesired harmful side effects. In this case, pros and cons must beweighed to decide whether the pharmaceutically active substance issuitable in the particular case. If the effect of the pharmaceuticallyactive substance inside a cell is predominantly beneficial for the wholeorganism, the cell is called a target cell. If the effect inside a cellis predominantly harmful for the whole organism, the cell is called anoff-target cell. In artificial systems such as cell cultures andbioreactors, target cells and off-target cells depend on the purpose andare defined by the user.

An effector moiety that is a polypeptide may be, e.g., a polypeptidethat recover a lost function, such as for instance enzyme replacement,gene regulating functions, or a toxin.

An embodiment is the first, fourth or sixth proteinaceous molecule ofthe invention, wherein the first, fourth or sixth proteinaceous moleculecomprises more than one saponin C, preferably 2, 3, 4, 5, 6, 8, 10, 16,32, 64 or 1-100 saponins, or any number of saponins therein between,such as 7, 9, 12 saponins, covalently bound directly to an amino-acidresidue of the first, fourth or sixth proteinaceous molecule, preferablyto a cysteine and/or to a lysine, and/or covalently bound via at leastone linker and/or via at least one cleavable linker and/or via at leastone polymeric or oligomeric scaffold L3, L7, preferably 1-8 of suchscaffolds or 2-4 of such scaffolds, wherein the at least one scaffold isoptionally based on a dendron, wherein 1-32 saponins such as 2, 3, 4, 5,6, 8, 10, 16, 32 saponins, or any number of saponins therein between,such as 7, 9, 12 saponins, are covalently bound to the at least onescaffold.

Table A1 and Scheme I and the above embodiments summarize a series ofsaponins C that have been identified for their endosomal escapeenhancing activity when contacted to mammalian cells, in particularhuman tumor cells, in free form together with a second molecule (e.g. aneffector moiety or effector molecule, payload, such as a toxin, anoligonucleotide). Indeed, in cell-based bioassays using human tumorcells it was established for the saponins tabulated in Table A1 andthose in Scheme I and in the various embodiments of the inventiondescribed herein, that under influence of these saponins, when bound tothe first, fourth or sixth proteinaceous molecule, a second molecule(effector moiety) such as a nucleic acid and/or a toxin such as aprotein toxin (e.g. one or more of the protein toxins listed in TableA5), bound to the second or third or fifth or seventh proteinaceousmolecule, is delivered into the cytosol with increased efficiency and/orefficacy, presumably through intracellular release from the (late)endosomes and lysosomes. That is to say, endosomal and/or lysosomalescape of such second molecules (effector moieties bound to a second orto a third or to a fifth or to a seventh proteinaceous molecule of theinvention), e.g. nucleic acids and/or toxins, is less efficient in theabsence of the saponin.

Surprisingly, the inventors now demonstrate that a water-soluble saponinfraction from Quillaja saponaria, comprising QS-21 and its familymembers QS-21A, QS-21 A-api, QS-21 A-xyl, QS-21B, QS-21 B-api, QS-21B-xyl, QS-7-xyl, QS-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862, QS-18and Quil-A, also exhibits the ability to potentiate a biological effectin vitro of e.g. a nucleic acid bound to a monoclonal antibody or aprotein toxin bound to a monoclonal antibody (examples of a secondand/or third and/or fifth and/or seventh proteinaceous molecule of theinvention comprising covalently bound oligonucleotide or payload such asa (protein) toxin), when administered to tumor cells of a mammalianspecies (human) in the form of a covalent conjugate comprising amonoclonal antibody (first, fourth, sixth proteinaceous molecule of theinvention), together with the second and/or third and/or fifth and/orseventh proteinaceous molecule comprising the effector moiety (theaforementioned second and/or third and/or fifth and/or seventhproteinaceous molecule) and the at least one glycoside such as the QS-21and its family member saponins encompassed by such QS-21 preparation(e.g. water soluble fraction of Quillaja saponaria), comprised by thefirst, fourth or sixth proteinaceous molecule as a covalent conjugate,wherein the effector molecule and the glycoside, e.g. saponin fractionof Quillaja saponaria, QS-21, SO1861, SA1641, GE1741, are covalentlybound to for example the proteinaceous molecules directly or via alinker or via a polymeric or oligomeric scaffold, either directly or viaat least one linker. Without wishing to be bound by any theory, theobserved stimulation or potentiation of for example antisense BNAmediated reduction of tumor-cell HSP27 expression (HSP27 gene silencing)in the presence of saponins derived from Quillaja saponaria in vitro may(also) relate to activation of the inflammasome in the tumor cell by thesaponins, for example resulting in tumor cell pyroptosis. The inventorsestablished that second and third and fifth and seventh proteinaceousmolecules conjugated to for example antisense BNA or dianthin orsaporin, exerted any anti-tumor cell activity in vitro at all orimproved anti-tumor cell activity when contacted with cells in bio-basedcell assays, when in the presence of the first or fourth or sixthproteinaceous molecule of the invention, comprising the saponin, andtargeted to the same (tumor) cells as the cell surface molecule targetedby the second and/or third and/or fifth and/or seventh proteinaceousmolecule, whereas in the absence of the first, fourth, sixthproteinaceous molecule and thus in the absence of saponin, no suchactivity towards the tumor cell was observed.

QS-21, and also the water-soluble saponins fraction comprising QS-21from Quillaja saponaria is already for a long time known and previouslyintensively applied for its immune-potentiating abilities, e.g. as anadjuvant in e.g. sub-unit vaccines. For example, QS-21 is applied in twophase III clinical trials with human patients, who were vaccinated witha sub-unit vaccine mixed with an adjuvant comprising QS-21(Glaxo-Smith-Kline, MAGRIT trial, DERMA study), wherein the sub-unit wasMAGE-A3 protein, which is specifically expressed and presented by tumorcells. The anti-tumor vaccinations, potentiated with QS-21, aimed forextension of disease-free survival of the cancer patients (melanoma;non-small cell lung cancer). In addition, QS-21 has been tested as anadjuvant in clinical trials for developing anti-cancer vaccinetreatment, for vaccines for HIV-1 infection, in development of a vaccineagainst hepatitis B, and for anti-malaria vaccine development usingQS-21 comprising adjuvants AS01 and AS02 of Glaxo-Smith-Kline. Previousstudies revealed an immune response elicited against MAGE-A3 peptidespresented at the cancer cell surface, under influence of the QS-21saponin comprising adjuvant (AS15; GSK). To the surprise of theinventors, the saponin fraction of Quillaja saponaria, and thus likelyQS-21 (as part of the water soluble saponin fraction of Quillajasaponaria) potentiates the anti-tumor cell activity of e.g. a payloadsuch as a protein toxin (dianthin), bound to the second, fifth, seventhproteinaceous molecule (e.g. the ligand EGF).

The inventors show that a tumor-cell targeting monoclonal antibodyprovided with covalently coupled antisense BNA such as BNA(HSP27), andcontacted with the tumor cells together with a first or fourthproteinaceous molecule of the invention with covalently coupled saponin(e.g. SO1861, QS-21), both the BNA and the saponin coupled to therespective antibody (e.g. cetuximab) of the first and third or fourthand fifth proteinaceous molecule via a cleavable bond is capable ofsilencing HSP27 in vivo in tumors, compared to control and compared toAOC (third and fifth proteinaceous molecule) only, without presence offirst or fourth proteinaceous molecule with coupled saponin.Co-administering an ADC or an antibody-oligonucleotide conjugate (AOC),such as an antibody-BNA conjugate, with a first or fourth proteinaceousmolecule with a saponin thus endows the ADC or AOC with anti-tumor cellactivity not seen with only the ADC or only the AOC at the same dose.Noteworthy, the AOC (the second or third or fifth proteinaceousmolecule) and the monoclonal antibody with covalently coupled saponin(first or fourth proteinaceous molecule) increase HSP27 expression intumor cells, when administered to tumor-bearing mice separately inseparate groups of mice, compared to a control group (vehicleadministered, only). Only co-administration of the AOC comprising theeffector moiety of the invention (second or third or fifth proteinaceousmolecule) and the first or fourth proteinaceous molecule with covalentlycoupled saponin, displays reduced HSP27 expression when compared tocontrols. The antisense BNA (HSP27) was BNA with oligo nucleic acidsequence 5′-GGCacagccagtgGCG-3′ according to Zhang et al. (2011) [YZhang, Z Qu, S Kim, V Shi, B Liao1, P Kraft, R Bandaru, Y Wu, LMGreenberger and ID Horak, Down-modulation of cancer targets using lockednucleic acid (LNA)-based antisense oligonucleotides withouttransfection, Gene Therapy (2011) 18, 326-333]. Noteworthy, to the bestof the knowledge of the inventors, BNA is designed for application as afree nucleic acid. The inventors are now the first to demonstrate thatthe antisense BNA can be covalently coupled through a (non-)cleavablelinker with a ligand or an antibody, in a way that gene-silencingactivity is retained in vitro and more importantly in vivo in the tumorcells of a tumor-bearing animal. This approach of providing BNA-basedAOCs opens new ways to administer targeted BNA to human (cancer)patients in need thereof.

The inventors disclose here that covalently coupling saponins such assaponins in the water-soluble fraction of Quillaja saponaria, QS-21,SA1641, SO1861, Table A1, Scheme I, to a first or fourth or sixthproteinaceous molecule, such as via a tri-functional linker, e.g. thetri-functional linker of Scheme II and Structure B, or via an oligomericor polymeric structure of a scaffold comprising covalently boundsaponins, results in improved cell toxicity exerted by the effectormoiety such as a toxin, comprised by the second and/or third and/orfifth and/or seventh proteinaceous molecule, under influence of thecovalently coupled saponin in the first, fourth or sixth proteinaceousmolecule.

According to the invention, typically the saponin is a saponin listed inTable A1, Scheme I. It has been proven beneficial for the activity ofthe saponin, e.g. the endosomal escape enhancing activity inside cellswhen the entry into the cell and the accumulation inside the cytosol ofan effector moiety covalently coupled to the second or third or fifth orseventh proteinaceous molecule, is considered, when the saponin iscovalently coupled to the first or fourth or sixth proteinaceousmolecule involving a hydrazone bond, and/or a hydrazide bond, and/or adisulphide bond. Such bond types readily cleave under the acidicconditions inside (late) endosomes and lysosomes of mammalian cells,e.g. human cells, and/or under the reductive conditions. Alternatively,the inventors also demonstrate that covalent coupling of saponin to thefirst, fourth or sixth proteinaceous molecule via a bond that is notreadily cleavable under the physiological conditions inside cells, e.g.(late) endosomes, lysosomes, cytosol, is also beneficial to thepotentiating activity of the saponin on the biological effect of e.g. aneffector moiety such as a nucleic acid (e.g. BNA silencing HSP27) and aproteinaceous toxin such as saporin. Throughout the application,including the claims, the term ‘cleavable linker’, ‘cleavable bond’,etc., is also referred to as ‘labile linker’ (‘L’) and ‘labile bond’,for example in the context of cleavage of such a bond or linker in the(late) endosome and/or lysosome when a conjugate of the invention, e.g.a first, fourth or sixth proteinaceous molecule optionally comprising ascaffold with saponins coupled to the first, fourth or sixthproteinaceous molecule through a linker and/or via the scaffold viahydrazone bonds or disulphide bonds, is referred to. For example, FIG.1-1, FIG. 6-2, FIG. 2-4 and FIG. 1-5 show the in vivo HSP27 genesilencing in human tumors in mice. The tumor-bearing mice were forexample treated with a first proteinaceous molecule consisting ofmonoclonal antibody with saponin bound thereto via a labile linker(hydrazone bond) according to the invention, whereas the thirdproteinaceous molecule comprised bound antisense BNA for silencing theHSP27 gene in the tumor cells, covalently coupled to the monoclonalantibody (same type as the first monoclonal antibody) via a disulphidebond. That is to say, without wishing to be bound by any theory, thehydrazone bond and the disulphide bond are cleaved in the (late)endosomes and/or lysosomes of the targeted tumor cells that express theepitope on the targeted cell-surface molecule, here the EGFR, at thecell surface, once the therapeutic combination of the invention isinternalized by e.g. endocytosis. Cleavage of the bonds likelycontributes to the endosomal escape enhancing activity of the saponinwhen the entry of the BNA from the endosome and/or lysosome into thecytosol is considered, although such cleavage is not a necessity forobserving the gene silencing effect of the combination of thecetuximab-SO1861 conjugate and the cetuximab-BNA conjugate of theinvention.

The skilled person will appreciate that a tri-functional linker is ascaffold of the invention suitable for covalently coupling one, two orthree saponin moieties. For the tri-functional linker covalent couplingof one or two saponin moieties is preferred. The second and/or thirdbinding site is for example suitable for covalent coupling aproteinaceous ligand such as the first, fourth or sixth proteinaceousmolecule. Typical proteinaceous ligands are EGF for targeting (tumor)cells expressing EGFR at the cell surface, and cytokines for targetingtumor cells or autoimmune cells. Moreover, the second or third bindingsite of the tri-functional linker is suitable for covalent coupling ofan immunoglobulin such as a monoclonal antibody, i.e. the first, fourthor sixth proteinaceous molecule for binding to a cell surface moleculesuch as a tumor cell surface molecule, preferably a tumor-cell specificmolecule, more preferably a tumor cell receptor that is specifically(over-)expressed at the surface of the tumor cell. Similarly, theimmunoglobulin, or any fragment(s) and/or domain(s) thereof whichencompass the binding specificity of the immunoglobulin, is suitable forbinding to a cell surface molecule such as a receptor, expressed at thesurface of an autoimmune cell. Thus, in an embodiment, the first, fourthor sixth proteinaceous molecule comprises the tri-functional linker,said linker comprises or consists of a covalently bound saponin, e.g.QS-21, SO1861, and the covalently bound binding site such as a celltargeting moiety such as a ligand or an antibody for (specific) bindingto a tumor cell, an auto-immune cell, a diseased cell, an aberrant cell,a non-healthy cell, a B-cell disease.

A first, fourth or sixth proteinaceous molecule according to theinvention thus comprises at least one saponin. With “at least one” inthis context is meant that the first, fourth or sixth proteinaceousmolecule comprises one saponin molecule but may also comprise a couple(e.g. two, three or four) of saponins or a multitude (e.g. 10, 20 or100) of saponins. Depending on the application, the first, fourth orsixth proteinaceous molecule may comprise a covalently bound scaffoldwith covalently bound saponins, wherein the scaffold may be designedsuch that it comprises a defined number of saponins. Preferably, afirst, fourth or sixth proteinaceous molecule according to the inventioncomprises a defined number or range of saponins, rather than a randomnumber. This is especially advantageous for drug development in relationto marketing authorization. A defined number in this respect means thata first, fourth or sixth proteinaceous molecule preferably comprises apreviously defined number of saponins. This is, e.g., achieved bydesigning a scaffold comprising a polymeric structure with a certainnumber of possible moieties for the saponin(s) to attach. Under idealcircumstances, all of these moieties are coupled to a saponin and thescaffold than comprises the prior defined number of saponins. It isenvisaged to offer a standard set of scaffolds, comprising, e.g., two,four, eight, sixteen, thirty-two, sixty-four, etc., saponins so that theoptimal number can be easily tested by the user according to his needs.An embodiment is the first, fourth or sixth proteinaceous molecule ofthe invention comprising the scaffold of the invention, wherein thesaponin is present in a defined range as, e.g., under non-idealcircumstances, not all moieties present in a polymeric structure bind asaponin. Such ranges may for instance be 2-4 saponin molecules perscaffold, 3-6 saponin molecules per scaffold, 4-8 saponin molecules perscaffold, 6-8 saponin molecules per scaffold, 6-12 saponin molecules perscaffold and so on. In such case, a first proteinaceous moleculecomprising a scaffold according to the invention thus comprises 2, 3 or4 saponins if the range is defined as 2-4.

The scaffold is fundamentally independent of the type of saponincovalently bound to the scaffold, the scaffold subsequently (insequential order) covalently coupled to the first, fourth or sixthproteinaceous molecule. Thus, first, fourth or sixth proteinaceousmolecule comprising the scaffold is the basis product for a new platformtechnology. Since the at least one covalently bound saponin mediatesintracellular delivery of the effector moiety bound to the second,third, fifth or seventh proteinaceous molecule, the scaffold technologyaccording to the invention is the first system known that mediatescontrolled intracellular effector moiety delivery by saponins. Thescaffold provides an optimized and functionally active unit that can belinked to the saponin(s) and to the binding site comprised by the first,fourth or sixth proteinaceous molecule, e.g. a ligand, an antibody,etc., at a single and defined position.

An embodiment is the first, fourth or sixth proteinaceous moleculecomprising a scaffold according to the invention, wherein the number ofmonomers of the polymeric or oligomeric structure is an exactly definednumber or range. Preferably, the polymeric or oligomeric structurecomprises structures such as poly(amines), e.g., polyethylenimine andpoly(amidoamine), or structures such as polyethylene glycol,poly(esters), such as poly(lactides), poly(lactams),polylactide-co-glycolide copolymers, poly(dextrin), or a peptide or aprotein, or structures such as natural and/or artificial polyaminoacids, e.g. poly-lysine, DNA polymers, stabilized RNA polymers or PNA(peptide nucleic acid) polymers, either appearing as linear, branched orcyclic polymer, oligomer, dendrimer, dendron, dendronized polymer,dendronized oligomer or assemblies of these structures, either sheer ormixed. Preferably, the polymeric or oligomeric structures arebiocompatible, wherein biocompatible means that the polymeric oroligomeric structure does not show substantial acute or chronic toxicityin organisms and can be either excreted as it is or fully degraded toexcretable and/or physiological compounds by the body's metabolism.Assemblies can be built up by covalent cross-linking or non-covalentbonds and/or attraction. They can therefore also form nanogels,microgels, or hydrogels, or they can be attached to carriers such asinorganic nanoparticles, colloids, liposomes, micelles or particle-likestructures comprising cholesterol and/or phospholipids. Said polymericor oligomeric structures preferably bear an exactly defined number orrange of coupling moieties for the coupling of glycoside molecules(and/or effector molecules and/or carrier molecules such as a ligand,monoclonal antibody or a fragment thereof). Preferably at least 50%,more preferably at least 75%, more preferably at least 85%, morepreferably at least 90%, more preferably at least 95%, more preferablyat least 98%, more preferably at least 99%, most preferably 100% of theexactly defined number or range of coupling moieties in the polymeric oroligomeric structure is occupied by a glycoside molecule in a scaffoldaccording to the invention.

Preferably, a dendron is a branched, clearly defined tree-like polymerwith a single chemically addressable group at the origin of the tree,called the focal point. A dendrimer is a connection of two or moredendrons at their focal point. A dendronized polymer is a connection ofthe focal point of one or more dendrons to a polymer. In a preferredembodiment, a scaffold according to the invention is provided, whereinthe polymeric or oligomeric structure comprises a linear, branched orcyclic polymer, oligomer, dendrimer, dendron, dendronized polymer,dendronized oligomer or assemblies of these structures, either sheer ormixed, wherein assemblies can be built up by covalent cross-linking ornon-covalent attraction and can form nanogels, microgels, or hydrogels,and wherein, preferably, the polymer is a derivative of a poly(amine),e.g., polyethylenimine and poly(amidoamine), and structures such aspolyethylene glycol, poly(esters), such as poly(lactids), poly(lactams),polylactide-co-glycolide copolymers, and poly(dextrin), and structuressuch as natural and/or artificial polyamino acids such as poly-lysine,or a peptide or a protein or DNA polymers, stabilized RNA polymers orPNA (peptide nucleic acid) polymers. Preferably, the polymeric oroligomeric structures are biocompatible.

An embodiment is the therapeutic combination of the invention or thetherapeutic combination for use according to the invention, wherein thefirst, fourth or sixth proteinaceous molecule comprises more than onecovalently bound saponin, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32, 64,128 or 1-100 saponins, or any number of saponins therein between, suchas 7, 9, 12 saponins.

An embodiment is the first, fourth or sixth proteinaceous molecule ofthe invention, wherein the at least one saponin is covalently bound tothe polymeric or oligomeric structure of the oligomeric or polymericscaffold via at least one cleavable linker according to the invention.

An embodiment is the first, fourth or sixth proteinaceous molecule ofthe invention, wherein the chemical group of the oligomeric or polymericscaffold, for covalently coupling of the oligomeric or polymericscaffold to the amino-acid residue of said first, fourth or sixthproteinaceous molecule, is a click chemistry group, preferably selectedfrom a tetrazine, an azide, an alkene or an alkyne, or a cyclicderivative of these groups, more preferably said chemical group is anazide.

An embodiment is the first, fourth or sixth proteinaceous molecule ofthe invention, wherein the polymeric or oligomeric structure of theoligomeric or polymeric scaffold comprises a linear, branched and/orcyclic polymer, oligomer, dendrimer, dendron, dendronized polymer,dendronized oligomer, a DNA, a polypeptide, poly-lysine, a poly-ethyleneglycol, or an assembly of these polymeric or oligomeric structures whichassembly is preferably built up by covalent cross-linking.

The inventors established that covalent coupling, preferably viacleavable bonds or linkers, of the saponin to the first, fourth or sixthproteinaceous molecule, according to any of the embodiments here above,provides efficient and cell-targeted potentiation of the activity of aneffector moiety bound to the second and to the third and to the fifthand to the seventh proteinaceous molecule, wherein the first and thirdand fourth and sixth proteinaceous molecules comprise the same first,fourth or sixth binding site and wherein the first (fourth, sixth) andsecond (fifth, seventh) proteinaceous molecules comprise a first (third,fourth, sixth) and second (fifth, seventh) binding site which aredifferent. Coupling saponin to a cysteine side chain or a lysine sidechain of the first, fourth or sixth proteinaceous molecule such as amonoclonal antibody, directly or via a linker, proved to be a beneficialway of specific and efficient delivery of effector-moiety potentiatingactivity inside the target cell, when also the effector moiety isdelivered in the same target cell by using the second and/or thirdand/or fifth and/or seventh proteinaceous molecule comprising the samefirst binding site as the first proteinaceous molecule when the third orfifth proteinaceous molecule is considered and comprising differentfirst and second, seventh binding sites respectively when the first andsecond, seventh proteinaceous molecules are considered.

To explain the invention in more detail, the process of cellular uptakeof substances (although the inventors do not wish to be bound by anytheory) and the used terminology in this invention is described. Theuptake of extracellular substances into a cell by vesicle budding iscalled endocytosis. Said vesicle budding can be characterized by (1)receptor-dependent ligand uptake mediated by the cytosolic proteinclathrin, (2) lipid-raft uptake mediated by the cholesterol-bindingprotein caveolin, (3) unspecific fluid uptake (pinocytosis), or (4)unspecific particle uptake (phagocytosis). All types of endocytosis runinto the following cellular processes of vesicle transport and substancesorting called the endocytic pathways. The endocytic pathways arecomplex and not fully understood. Without wishing to be bound by anytheory, organelles may be formed de novo and mature into the nextorganelle along the endocytic pathway. It is however, now hypothesizedthat the endocytic pathways involve stable compartments that areconnected by vesicular traffic. A compartment is a complex,multifunctional membrane organelle that is specialized for a particularset of essential functions for the cell. Vesicles are considered to betransient organelles, simpler in composition, and are defined asmembrane-enclosed containers that form de novo by budding from apreexisting compartment. In contrast to compartments, vesicles canundergo maturation, which is a physiologically irreversible series ofbiochemical changes. Early endosomes and late endosomes represent stablecompartments in the endocytic pathway while primary endocytic vesicles,phagosomes, multivesicular bodies (also called endosome carriervesicles), secretory granules, and even lysosomes represent vesicles.The endocytic vesicle, which arises at the plasma membrane, mostprominently from clathrin-coated pits, first fuses with the earlyendosome, which is a major sorting compartment of approximately pH 6.5.A large part of the cargo and membranes internalized are recycled backto the plasma membrane through recycling vesicles (recycling pathway).Components that should be degraded are transported to the acidic lateendosome (pH lower than 6) via multivesicular bodies. Lysosomes arevesicles that can store mature lysosomal enzymes and deliver them to alate endosomal compartment when needed. The resulting organelle iscalled the hybrid organelle or endolysosome. Lysosomes bud off thehybrid organelle in a process referred to as lysosome reformation. Lateendosomes, lysosomes, and hybrid organelles are extremely dynamicorganelles, and distinction between them is often difficult. Degradationof an endocytosed molecule occurs inside an endolysosome or lysosome.Endosomal escape is the active or passive release of a substance fromthe inner lumen of any kind of compartment or vesicle from the endocyticpathway, preferably from clathrin-mediated endocytosis, or recyclingpathway into the cytosol. Endosomal escape thus includes but is notlimited to release from endosomes, endolysosomes or lysosomes, includingtheir intermediate and hybrid organelles.

Unless specifically indicated otherwise and in particular when relatingto the endosomal escape mechanism of the glycoside molecule such as thesaponin of the invention, whenever the word “endosome” or “endosomalescape” is used herein, it also includes the endolysosome and lysosome,and escape from the endolysosome and lysosome, respectively. Afterentering the cytosol, said substance might move to other cell units suchas the nucleus.

In formal terms, a glycoside is any molecule in which a sugar group isbound through its anomeric carbon to another group via a glycosidicbond. Glycoside molecules, such as saponins, in the context of theinvention are such molecules that are further able to enhance the effectof an effector moiety, without wishing to be bound by any theory, inparticular by facilitating the endosomal escape of the effector moiety.Without wishing to be bound by any theory, the glycoside molecules(saponins, such as those listed in Table A1) interact with the membranesof compartments and vesicles of the endocytic and recycling pathway andmake them leaky for said effector moieties resulting in augmentedendosomal escape. With the term “the scaffold is able to augmentendosomal escape of the effector moiety” is meant that the at least onesaponin (glycoside molecule), which is coupled to the polymeric oroligomeric structure of the scaffold, is able to enhance endosomalescape of an effector moiety when both molecules are within an endosome,e.g. a late endosome, optionally and preferably after the at least oneglycoside such as a saponin is released from the first, fourth, sixthproteinaceous molecule such as from a linker or polymeric or oligomericstructure comprised by said first, fourth, sixth proteinaceous molecule,e.g., by cleavage of a cleavable bond between the at least one glycoside(saponin) and the first, fourth, sixth proteinaceous molecule (forexample via a polymeric or oligomeric structure of a scaffold and/or viaa linker). Even though a bond between the at least one glycoside such asa saponin according to the invention and the first, fourth, sixthproteinaceous molecule, optionally via a linker or a scaffold, may be a“stable bond”, that does not mean that such bond cannot be cleaved inthe endosomes by, e.g., enzymes. For instance, the glycoside or saponin,optionally together with a linker or a part of the oligomeric orpolymeric structure of a scaffold, may be cleaved off from the remaininglinker fragment or oligomeric or polymeric structure. It could, forinstance be that a protease cuts a (proteinaceous) linker orproteinaceous polymeric structure, e.g., albumin, thereby releasing theat least one glycoside, saponin. It is, however, preferred that theglycoside molecule (preferably saponin) is released in an active form,preferably in the original form that it had before it was (prepared tobe) coupled to the first, fourth, sixth proteinaceous moleculeoptionally via a linker and/or an oligomeric or polymeric scaffold; thusthe glycoside (saponin) has its natural structure after such cleavage orthe glycoside (saponin) has (part of) a chemical group or linker boundthereto, after such cleavage, while glycoside biological activity(saponin biological activity), e.g. endosomal/lysosomal escape enhancingactivity towards an effector moiety present in the same endosome orlysosome, is maintained or restored upon said cleavage of the bondbetween the glycoside (saponin) and the carrier molecule, i.e. thefirst, fourth, sixth proteinaceous molecule optionally comprising alinker and/or a scaffold of the invention. With regard to the presentinvention the term “stable” with respect to bonds between e.g. saponinsand amino-acid residues of the first, fourth, sixth proteinaceousmolecule, a linker, a polymeric or oligomeric structures (of thescaffold), ligands, (monoclonal) immunoglobulins or binding domains or-fragments thereof, and/or effectors (effector moieties, effectormolecules), is meant that the bond is not readily broken or at least notdesigned to be readily broken by, e.g., pH differences, saltconcentrations, or UV-light, reductive conditions. With regard to thepresent invention the term “cleavable” with respect to bonds betweene.g. saponins and the first, fourth, sixth proteinaceous molecule,linkers, amino-acid residues, polymeric or oligomeric structures of thescaffold, ligands, antibodies and/or effectors, is meant that the bondis designed to be readily broken by, e.g., pH differences, saltconcentrations, under reductive conditions, and the like. The skilledperson is well aware of such cleavable bonds and how to prepare them.

Before the present invention one of the major hurdles of introducingADCs and AOCs on the market was the small therapeutic window: atherapeutically effective dose of an ADC or an AOC is accompanied with(unacceptable) side effects, hampering development and implication intreatment of patients with the ADCs. By the application of the first,fourth or sixth proteinaceous molecule of the invention it has nowbecome possible to guide one or multiple glycoside molecules (saponin)to a (target) cell, together with the ADC carrying a payload or togetherwith a (monoclonal) antibody conjugated with an oligonucleotide such asa BNA according to the invention (i.e. a particular second or third orfifth or seventh proteinaceous molecule of the invention). Inparticular, it was previously not possible to specifically guide aneffector moiety of a second or third or fifth or seventh proteinaceousmolecule and a (predefined, controllable) particular number or range ofglycoside molecules (saponins) per effector moiety at the same time tothe cytosol of cells, such as via the endocytic pathway of a cell.

A solution provided for by the invention comprises the covalent bindingof at least one saponin to the first or fourth or sixth proteinaceousmolecule. A further solution provided for by the invention comprises(first) polymerizing the glycoside molecules (saponins) using anoligomeric or polymeric scaffold, and providing the first or fourth orsixth proteinaceous molecule with a cluster of covalently boundsaponins, enabling re-monomerization of the one or more saponins at theintracellular site where the mode of action of the saponin is desired,e.g. after endocytosis. “Polymerizes” in this context means thereversible and/or irreversible multiple conjugation of saponin moleculesto the first, fourth, sixth proteinaceous molecule, either via linker,or directly or via a polymeric or oligomeric structure to form ascaffold or the reversible and/or irreversible multiple conjugation of(modified) saponins thereby forming a polymeric or oligomeric structureto form a scaffold. “Re-monomerization” in this context means thecleavage of the saponins from the first, fourth, sixth proteinaceousmolecule, from the linker linking the saponin(s) to the first, fourth,sixth proteinaceous molecule or from the scaffold, for example afterendocytosis, and regaining the (native) chemical state of the unboundsaponins, which unbound saponins may or may not comprise additionalchemical groups such as a chemical group for linking the saponin to alinker, an amino-acid residue of the first proteinaceous molecule or tothe scaffold, and/or a (chemical) linker bound to a chemical group ofthe saponin such as an aldehyde group or carboxylic acid group. Due tothe complex chemistry of the saponins for example the ‘polymerization’of saponins at a scaffold or other linking linker and their“re-monomerization” at a desired location such as intracellularly e.g.after endocytosis, was a challenging task. In particular, the chemicalreactions used for providing the linkers and the scaffold comprisingcovalently linked glycosides for covalent binding to the first, fourth,sixth proteinaceous molecule, e.g. triterpenoid saponins (polymerizationof the glycosides), normally occur in water-free organic solvents, butsaponins and for example biocompatible polymers applied as a scaffoldfor bearing bound saponins, are water-soluble molecules. The chemicalproperties of the unmodified saponin further prohibited polymerizationby itself and, one other possible solution, to bind multiple saponins(directly) to the effector molecule was estimated not to be verypromising, as an effector molecule (drug, toxin, polypeptide orpolynucleotide) does typically not provide sufficient binding sites andbecause the coupling product would become quite heterogeneous and/orcoupling biologically active molecules such as a saponin and e.g. apeptide, a toxin, a nucleic acid together bears the risk for influencingand hampering the activity of one or even both molecules bound togetherin such saponin-comprising conjugate. Further, there was a considerablerisk that the effector moiety comprised by the second or third or fifthor seventh proteinaceous molecule loses its function after coupling of asaponin to the e.g. ADC or antibody-oligonucleotide conjugate (AOC).Embodiments of the present invention solves at least one of thesedrawbacks.

An aspect of the invention relates to a composition comprising the firstor fourth or sixth proteinaceous molecule of the invention and thesecond, third, fifth or seventh proteinaceous molecule of the invention.

An aspect of the invention relates to a composition comprising thefirst, fourth or sixth proteinaceous molecule of the invention and thethird or fifth proteinaceous molecule of the invention.

An embodiment is the composition comprising the first, fourth or sixthproteinaceous molecule of the invention and the second, or seventhproteinaceous molecule of the invention, or is the compositioncomprising the first, fourth or sixth proteinaceous molecule of theinvention and the third or fifth proteinaceous molecule of theinvention, wherein the effector moiety that is comprised by the second,fifth or seventh proteinaceous molecule or by the third proteinaceousmolecule is any one of the effector moieties according to the invention,preferably a BNA.

An aspect of the invention relates to a composition comprising thefirst, fourth or sixth proteinaceous molecule of the invention and anyone or more of an oligonucleotide, a nucleic acid and a xeno nucleicacid, preferably selected from at least one of a vector, a gene, a cellsuicide inducing transgene, deoxyribonucleic acid (DNA), ribonucleicacid (RNA), anti-sense oligonucleotide (ASO, AON), short interfering RNA(siRNA), microRNA (miRNA), DNA aptamer, RNA aptamer, mRNA, mini-circleDNA, peptide nucleic acid (PNA), phosphoramidate morpholino oligomer(PMO), locked nucleic acid (LNA), bridged nucleic acid (BNA),2′-deoxy-2′-fluoroarabino nucleic acid (FANA), 2′-O-methoxyethyl-RNA(MOE), 2′-O,4′-aminoethylene bridged nucleic acid, 3′-fluoro hexitolnucleic acid (FHNA), a plasmid, glycol nucleic acid (GNA) and threosenucleic acid (TNA), or a derivative thereof, more preferably a BNA, forexample a BNA for silencing HSP27 protein expression (antisenseBNA(HSP27)).

An effector molecule, or effector moiety, in the context of thisinvention is any substance that affects the metabolism of a cell byinteraction with an intracellular effector molecule target, wherein thiseffector molecule target is any molecule or structure inside cellsexcluding the lumen of compartments and vesicles of the endocytic andrecycling pathway but including the membranes of these compartments andvesicles. Said structures inside cells thus include the nucleus,mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus,other transport vesicles, the inner part of the plasma membrane and thecytosol. Cytosolic delivery of an effector moiety in the context of theinvention preferably means that the effector moiety is able to escapethe endosome (and/or lysosome), which, as defined previously, alsoincludes escaping the endolysosome and the lysosome, and is preferablyable to reach the effector moiety target as described herein. Theinvention also encompasses a new type of molecule, referred to asscaffold that serves to bring both an effector moiety and at least oneglycoside molecule such as a saponin of the invention in an endosome atthe same time in a pre-defined ratio, when the effector moiety iscomprised by the second or third or fifth or seventh proteinaceousmolecule of the invention and the saponin is comprised by the first,fourth or sixth proteinaceous molecule. Within the context of thepresent invention, the polymeric or oligomeric structure of the scaffoldis a structurally ordered formation such as a polymer, oligomer,dendrimer, dendronized polymer, or dendronized oligomer or it is anassembled polymeric structure such as a hydrogel, microgel, nanogel,stabilized polymeric micelle or liposome, but excludes structures thatare composed of non-covalent assemblies of monomers such ascholesterol/phospholipid mixtures. The terms “polymer, oligomer,dendrimer, dendronized polymer, or dendronized oligomer” have theirordinary meaning. In particular a polymer is a substance which has amolecular structure built up chiefly or completely from a large numberof equal or similar units bonded together and an oligomer is a polymerwhose molecules consist of relatively few repeating units. There is noconsensus about one specific cut-off for “many” and “a few” as used inthe above definition of polymer and oligomer, respectively. However, asthe scaffold may comprise a polymeric or an oligomeric structure, orboth, the full range of numbers of similar units bonded together appliesto such structure, i.e. from 2 monomeric units to 100 monomeric units,1000 monomeric units, and more. A structure comprising 5 or less, forinstance maybe called an oligomeric structure, whereas a structurecomprising 50 monomeric units maybe called a polymeric structure. Astructure of 10 monomeric units maybe called either oligomeric orpolymeric. A scaffold as defined herein, further comprises at least oneglycoside molecule such as a saponin of the invention. A scaffoldpreferably includes a polymeric or oligomeric structure such as poly- oroligo(amines), e.g., polyethylenimine and poly(amidoamine), andbiocompatible structures such as polyethylene glycol, poly- oroligo(esters), such as poly(lactids), poly(lactams),polylactide-co-glycolide copolymers, and poly(dextrin), poly- oroligosaccharides, such as cyclodextrin or polydextrose, and poly- oroligoamino acids, such as poly-lysine or a peptide or a protein, or DNAoligo- or polymers. An assembled polymeric structure as defined hereincomprises at least one scaffold and, optionally, other individualpolymeric or oligomeric structures. Other individual polymeric oroligomeric structures of said assembly may be (a) scaffolds (thuscomprising at least one glycoside molecule such as a saponin of theinvention), (b) functionalized scaffolds (thus comprising at least oneglycoside molecule such as a saponin, and a ligand, antibody, etc. asthe first proteinaceous molecule, (c) polymeric or oligomeric structureswithout a glycoside molecule such as a saponin of the invention (SeeTable A1 for example), without a ligand, antibody, etc., as the firstproteinaceous molecule. A functionalized assembled polymeric structureis an assembled polymeric structure that contains (a) at least onefunctionalized scaffold or (b) at least one scaffold and at least onepolymeric structure comprising at least one ligand, antibody, etc. asthe first proteinaceous molecule. Polymeric or oligomeric structureswithin an assembled polymeric structure that do not comprise any of theabove mentioned molecules (i.e. no glycosides such as saponins, no firstproteinaceous molecule such as ligands, antibodies) are in particularadded as structural components of the assembled structures, which helpto build up or to stabilize the assembled structure(“glue-like”).Without wishing to be bound by any theory, the acidicenvironment seems to be a prerequisite for the synergistic actionbetween glycoside (saponin) and effector moiety.

Whether or not a first, fourth or sixth proteinaceous moleculecomprising saponins, either or not further comprising one or more(cleavable) linkers and/or optionally a scaffold, is able to disturb theacidic environment and inhibit the endosomal escape function of the atleast one glycoside (saponin) can be easily determined with an assayknown in the art. The inhibition is described as “fold amount increasesof glycoside necessary to induced 50% cell killing”. It is preferredthat the scaffold does not lead to an increase that is at least theincrease in glycoside molecules (saponins) necessary to obtain 50% cellkilling observed when using Chloroquine as a positive control.Alternatively, and preferably, the first, fourth, sixth proteinaceousmolecule comprising saponins, either or not further comprising one ormore (cleavable) linkers and/or optionally a scaffold does not lead toan at least 4-fold increase of glycoside molecules to induce 50% cellkilling, more preferably does not lead to an at least 2-fold increase.The fold increase is to be measured in assay, wherein Chloroquine, as apositive control, induces a 2-fold increase in glycoside amount,preferably saponin amount wherein the saponin is any one or more of thesaponins of the invention (see Table A1, Scheme I, previous embodiments)to observe 50% cell killing.

With the term “improving or enhancing an effect of an effector moiety”is meant that the glycoside molecule, preferably a saponin of theinvention, increases the functional efficacy of that effector moiety(e.g. the therapeutic index of a toxin or a drug or an oligonucleotidesuch as a BNA; the metabolic efficacy of a modifier in biotechnologicalprocesses; the transfection efficacy of genes in cell culture researchexperiments), preferably by enabling or improving its target engagement.Acceleration, prolongation, or enhancement of antigen-specific immuneresponses are preferably not included. Therapeutic efficacy includes butis not limited to a stronger therapeutic effect, preferably with lowerdosing and/or with less side effects. “Improving an effect of aneffector moiety” can also mean that an effector moiety, which could notbe used because of lack of effect (and was e.g. not known as being aneffector moiety), becomes effective when used in combination with thepresent invention. Any other effect, which is beneficial or desired andcan be attributed to the combination of effector moiety and the secondor third proteinaceous molecule, as provided by the invention isconsidered to be “an improved effect”. In an embodiment, the scaffoldcomprising bound saponin(s) and comprised by the first, fourth or sixthproteinaceous molecule enhances an effect of the effector moietycomprised by the second, third, fifth or seventh proteinaceous moleculewhich effect is intended and/or desired. In case of a first, fourth orsixth proteinaceous molecule comprising saponin bound to a proteinaceousscaffold, the proteinaceous polymeric structure of the scaffold as suchmay have, for instance, an effect on colloid osmotic pressure in theblood stream. If such effect is not the intended or desired effect ofsuch a functionalized scaffold comprised by the first, fourth or sixthproteinaceous molecule, the proteinaceous structure of the scaffold isnot an effector moiety as defined in the invention. Or, for instance incase of a DNA- or RNA-based scaffold carrying bound saponins andcomprised by the first proteinaceous molecule, parts of that DNA or RNAmay have an (unintended) function, e.g., by interfering with expression.If such interference is not the intended or desired effect of theultimate functionalized scaffold, the DNA- or RNA polymeric structure ofthe scaffold is not the effector moiety as defined in the invention.

A number of preferred features can be formulated for endosomal escapeenhancers comprised by the first, fourth or sixth proteinaceousmolecule, i.e. a glycoside or saponin, preferably a saponin according tothe invention: (1) they are preferably not toxic and do not invoke animmune response, (2) they preferably do not mediate the cytosolic uptakeof the effector moiety into off-target cells, (3) their presence at thesite of action is preferably synchronized with the presence of theeffector moiety, (4) they are preferably biodegradable or excretable,and (5) they preferably do not substantially interfere with biologicalprocesses of the organism unrelated to the biological activity of theeffector molecule with which the endosomal escape enhancer is combinedwith, e.g. interact with hormones. Examples of glycoside molecules suchas saponins of the invention that fulfill the before mentioned criteria,at least to some extent, are bisdesmosidic triterpenes, preferablybisdesmosidic triterpene saponins, such as SO1861, SA1641, QS-21,GE1741, and the saponins in Table A1, Scheme I.

An aspect of the invention relates to an antibody-drug conjugate or anantibody-oligonucleotide conjugate or a ligand-drug conjugate comprisingthe first, fourth or sixth proteinaceous molecule of the invention andan effector moiety.

As said before, the at least one saponin that is comprised by the first,fourth or sixth proteinaceous molecule according to the inventionincreases the efficacy of at least current and new effector moieties asdefined in this invention. Potential side-effects will be decreased dueto lowering of dosing of the effector moiety comprised by the second orthird or fifth or seventh proteinaceous molecule, without lowering theefficacy. Therefore, the invention provides a first, fourth or sixthproteinaceous molecule according to the invention for use in medicine orfor use as a medicament. Thus, an aspect of the invention relates to afirst, fourth or sixth proteinaceous molecule according to theinvention, the first, fourth or sixth proteinaceous molecule comprisingat least a saponin, for use as a medicament. Also provided is the use ofa first, fourth or sixth proteinaceous molecule according to theinvention for manufacturing a medicament. Especially cancer medicines,and in particular the classical chemotherapy medicaments, are notoriousfor their side effects. Because of targeting and synchronization in timeand place of both the pharmaceutically active substance comprised by thesecond or third or fifth or seventh proteinaceous molecule and thesaponin comprised by the first, fourth or sixth proteinaceous molecule,since the first and third, and/or the fourth and fifth, proteinaceousmolecule bear the same binding site for the same epitope on the samecell-surface molecule, or since the first and second, or sixth andseventh, proteinaceous molecule bear different binding sites fordifferent first and second, or sixth and seventh, epitopes on the firstand second, or on the sixth and seventh, cell-surface moleculesrespectively, a therapeutic combination according to the invention isespecially valuable for use as a medicament, in particular for use in amethod of treating cancer. The invention thus provides a therapeuticcombination according to the invention or a first, fourth or sixthproteinaceous molecule of the invention for use in a method of treatingcancer. The invention also provides a therapeutic combination accordingto the invention or a first, fourth or sixth proteinaceous molecule ofthe invention for use in a method of treating acquired or hereditarydisorders, in particular monogenic deficiency disorders. The therapeuticcombination thus comprises the first and second proteinaceous moleculeand/or comprises the first and third proteinaceous molecule, and/or thefourth and fifth proteinaceous molecule, and/or the sixth and seventhproteinaceous molecule. Thus, an aspect of the invention relates to atherapeutic combination according to the invention, wherein the secondor third or fifth or seventh proteinaceous molecule comprises acovalently bound effector moiety, for use in a method for the treatmentof a cancer or an auto-immune disease.

A further application of the first, second and third, fourth, fifth,sixth, seventh proteinaceous molecules of the invention in medicine isthe substitution of intracellular enzymes in target cells that producethese enzymes in insufficient amount or insufficient functionality. Theresulting disease might be hereditary or acquired. In most cases, onlysymptomatic treatment is possible and for a number of rare diseases,insufficient treatment options lead to a shortened life span ofconcerned patients. An example for such a disease is phenylketonuria,which is an inborn error of metabolism that results in decreasedmetabolism of the amino acid phenylalanine. The disease is characterizedby mutations in the gene for the hepatic enzyme phenylalaninehydroxylase. Phenylketonuria is not curable to date. The incidence isapproximately 1:10,000 with the highest known incidence in Turkey with1:2,600. A second or third or fifth or seventh proteinaceous molecule,preferably an antibody, with bound phenylalanine hydroxylase or with abound polynucleotide that encodes phenylalanine hydroxylase can be usedto target liver cells by use of a suitable specific antibody, and tosubstitute the defect enzyme in hepatocytes. This is one example of useof the therapeutic combination of the invention comprising a first,fourth or sixth proteinaceous molecule with a saponin bound thereto anda second or third or fifth or seventh proteinaceous molecule with theenzyme or the oligonucleotide bound thereto according to the inventionfor substitution or gene therapy. In a preferred embodiment, atherapeutic combination according to the invention for use in a methodof gene therapy or substitution therapy is provided.

The present invention also provides a method of treating cancer, themethod comprising administering a medicament comprising a therapeuticcombination according to the invention to a patient in need thereof,preferably administering an effective dose of said medicament to apatient in need thereof, preferably a human cancer patient.

Considerations concerning forms suitable for administration are known inthe art and include toxic effects, solubility, route of administration,and maintaining activity. For example, pharmacological compositionsinjected into the bloodstream should be soluble.

Suitable dosage forms, in part depend upon the use or the route ofentry, for example transdermal or by injection. Such dosage forms shouldallow the compound to reach a target cell whether the target cell ispresent in a multicellular host. Other factors are known in the art, andinclude considerations such as toxicity and dosage form which retard thecompound or composition from exerting its effect.

An embodiment is the combination of an endosomal escape enhancingconjugate according to the invention, comprising the first, fourth orsixth proteinaceous molecule comprising at least one covalently boundsaponin, and a binding moiety, wherein the binding moiety comprises atleast one effector moiety, the binding moiety being the second or thirdor fifth or seventh proteinaceous molecule comprising the bound effectormoiety, wherein the endosomal escape enhancing conjugate and the bindingmoiety are, independently from one another, able to specifically bind toa target cell-specific surface molecule or structure, thereby inducingreceptor-mediated endocytosis of a complex of the endosomal escapeenhancing conjugate and the target cell-specific surface molecule, andof the complex of the binding moiety and the target cell-specificsurface molecule, wherein the endosomal escape enhancing conjugate andthe binding moiety can bind to the same target cell-specific surfacemolecule via their same binding site, or wherein the endosomal escapeenhancing conjugate and the binding moiety can bind to the differenttarget cell-specific surface molecules via their different bindingsites. An embodiment is the combination according to the invention,wherein the endosomal escape enhancing conjugate is able to compete withthe binding moiety for binding to the target cell-specific surfacemolecule or structure. An embodiment is the combination according to theinvention, wherein the endosomal escape enhancing conjugate and thebinding moiety are, independently from one another, able to specificallybind to the same epitope, or to a different epitope. An embodiment isthe combination for use in a method for the treatment of an aberrancysuch as a cancer according to the invention, wherein said endosomalescape enhancing conjugate and said binding moiety are to beadministered concomitant or sequentially, preferably concomitant.

An aspect of the invention relates to a kit comprising a first containercontaining an endosomal escape enhancing conjugate according to theinvention (i.e. the first, fourth or sixth proteinaceous molecule) and asecond container containing a binding moiety according to the invention(i.e. the second and/or third and/or fifth and/or seventh proteinaceousmolecule), the kit further comprising instructions for using the bindingmolecules (i.e. the therapeutic combination comprising the first andsecond or the first and third or the fourth and fifth or the sixth andseventh pharmaceutical compositions).

It is part of the invention that the therapeutic combination, the firstpharmaceutical composition, the first proteinaceous molecule, the secondor third pharmaceutical composition or the second or third proteinaceousmolecule of the invention is further combined with a covalent conjugate(complex) of a binding molecule or a binding moiety and a saponin, or isfurther combined with a pharmaceutical compound, an antibody, etc.,therewith providing a composition comprising three or more enhancers,pharmaceutically active ingredients, etc., e.g. a conjugate of theinvention (e.g. a first proteinaceous molecule and/or a second or thirdproteinaceous molecule) combined with a binding moiety complexed with aneffector molecule, further combined with a pharmaceutical, which iseither or not linked to a saponin, and which is either or not coupled toa ligand such as a targeting immunoglobulin, a domain or a fragmentthereof. Furthermore, an embodiment is the therapeutic combination, thefirst pharmaceutical composition, the first proteinaceous molecule, thesecond or third pharmaceutical composition or the second or thirdproteinaceous molecule of the invention, wherein the second or thirdproteinaceous molecule is provided with two or more effector moietiessuch as a toxin or immunotoxin, wherein the two or more effectormoieties are the same or different.

TABLE A1 Saponins displaying (late) endosomal/lysosomal escape enhancingactivity, and saponins comprising a structure reminiscent to suchsaponins displaying (late) endosomal/lysosomal escape enhancing activityCarbohydrate substituent Saponin Name Aglycon core at the C-3beta-OHgroup Carbohydrate substituent at the C-28-OH group NP-005236 2alpha-GlcA- Glc/Gal- Hydroxyoleanolic acid AMA-1 16alpha- Glc-Rha-(1→2)-[Xyl-(1→4)]-Rha- Hydroxyoleanolic acid AMR 16alpha- Glc-Rha-(1→2)-[Ara-(1→3)-Xyl-(1→4)]-Rha- Hydroxyoleanolic acid alpha-HederinHederagenin (23- Rha-(1→2)-Ara- — Hydroxyoleanolic acid) NP-01267216alpha,23- Ara/Xyl-(1→4)-Rha/Fuc- Ara/Xyl- Dihydroxyoleanolic(1→2)-Glc/Gal-(1→2)- acid Rha/Fuc-(1→2)-GlcA- NP-017777 GypsogeninGal-(1→2)-[Xyl-(1→3)]-GlcA- Xyl-(1→4)-Rha-(1→2)-[R-(→4)]-Fuc- (R = 4E-Methoxycinnamic acid) NP-017778 Gypsogenin Gal-(1→2)-[Xyl-(1→3)]-GlcA-Xyl-(1→4)-Rha-(1→2)-[R-(→4)]-Fuc- (R = 4Z- Methoxycinnamic acid)NP-017774 Gypsogenin Gal-(1→2)-[Xyl-(1→3)]-GlcA-Xyl-(1→4)-[Gal-(1→3)]-Rha-(1→2)-4-OAc- Fuc- NP-018110^(c), GypsogeninGal-(1→2)-[Xyl-(1→3)]-GlcA- Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-3,4-di-NP-017772^(d) OAc-Fuc- NP-018109 Gypsogenin Gal-(1→2)-[Xyl-(1→3)]-GlcA-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[R-(→4)]- 3-OAc-Fuc- (R =4E-Methoxycinnamic acid) NP-017888 GypsogeninGal-(1→2)-[Xyl-(1→3)]-GlcA- Glc-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-4-OAc-Fuc- NP-017889 Gypsogenin Gal-(1→2)-[Xyl-(1→3)]-GlcA-Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-4-OAc-Fuc- NP-018108 GypsogeninGal-(1→2)-[Xyl-(1→3)]-GlcA- Ara/Xyl-(1→3)-Ara/Xyl-(1→4)-Rha/Fuc-(1→2)-[4-OAc-Rha/Fuc-(1→4)]-Rha/Fuc- SA1641^(a), GypsogeninGal-(1→2)-[Xyl-(1→3)]-GlcA- Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Qui- AEX55^(b) (1→4)]-Fuc- NP-017674 Quillaic acid Gal-(1→2)-[Xyl-(1→3)]-GlcA-Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha- (1→2)-Fuc- NP-017810 Quillaic acidGal-(1→2)-[Xyl-(1→3)]-GlcA- Xyl-(1→4)-[Gal-(1→3)]-Rha-(1→2)-Fuc- AG1Quillaic acid Gal-(1→2)-[Xyl-(1→3)]-GlcA-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-Fuc- NP-003881 Quillaic acidGal-(1→2)-[Xyl-(1→3)]-GlcA- Ara/Xyl-(1→4)-Rha/Fuc-(1→4)-[Glc/Gal-(1→2)]-Fuc- NP-017676 Quillaic acid Gal-(1→2)-[Xyl-(1→3)]-GlcA-Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha- (1→2)-[R-(→4)]-Fuc- (R =5-O-[5-O-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl- octanoic acid) NP-017677Quillaic acid Gal-(1→2)-[Xyl-(1→3)]-GlcA-Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R-(→4)]- Fuc- (R =5-O-[5-O-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl- octanoic acid) NP-017706Quillaic acid Gal-(1→2)-[Xyl-(1→3)]-GlcA-Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Rha- (1→3)]-4-OAc-Fuc- NP-017705 Quillaicacid Gal-(1→2)-[Xyl-(1→3)]-GlcA- Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-(1→2)-[Rha-(1→3)]-4-OAc-Fuc- NP-017773 Quillaic acidGal-(1→2)-[Xyl-(1→3)]-GlcA- 6-OAc-Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3-OAc-Rha-(1→3)]-Fuc- NP-017775 Quillaic acid Gal-(1→2)-[Xyl-(1→3)]-GlcA-Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3-OAc-- Rha-(1→3)]-Fuc- SA1657 Quillaicacid Gal-(1→2)-[Xyl-(1→3)]-GlcA- Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Qui-(1→4)]-Fuc- AG2 Quillaic acid Gal-(1→2)-[Xyl-(1→3)]-GlcA-Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-[Qui- (1→4)]-Fuc- SO1861 Quillaic acidGal-(1→2)-[Xyl-(1→3)]-GlcA- Glc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuc- GE1741 Quillaic acid Gal-(1→2)-[Xyl-(1→3)]-GlcA-Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[3,4-di-OAc- Qui-(1→4)]-Fuc- SO1542Quillaic acid Gal-(1→2)-[Xyl-(1→3)]-GlcA-Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)-Fuc- SO1584 Quillaic acidGal-(1→2)-[Xyl-(1→3)]-GlcA- 6-OAc-Glc-(1→3)-[Xyl-(1→4)]-Rha-(1→2)- Fuc-SO1658 Gypsogenin Gal-(1→2)-[Xyl-(1→3)]-GlcA-Glc-(1→3)-[Xyl-(1→3)-Xyl-(1→4)]-Rha- (1→2)-Fuc- SO1674 Quillaic acidGal-(1→2)-[Xyl-(1→3)]-GlcA- Glc-(1→3)-[Xyl-(1→3)-Xyl-(1→4)]-Rha-(1→2)-Fuc- SO1832 Quillaic acid Gal-(1→2)-[Xyl-(1→3)]-GlcA-Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)- 4-OAc-Qui-(1→4)]-Fuc- QS-7(also referred Quillaic acid Gal-(1→2)-[Xyl-(1→3)]-GlcA-Api/Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha- to as QS1861)(1→2)-[Rha-(1→3)]-4OAc-Fuc- QS-7 api (also Quillaic acidGal-(1→2)-[Xyl-(1→3)]-GlcA- Api-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha-referred to as (1→2)-[Rha-(1→3)]-4OAc-Fuc- QS1862) QS-17 Quillaic acidGal-(1→2)-[Xyl-(1→3)]-GlcA- Api/Xyl-(1→3)-Xyl-(1-4)-[Glc-(1→3)]-Rha-(1→2)-[R-(→4)]-Fuc- (R = 5-O-[5-O-Rha-(1→2)-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy- 6-methyl-octanoic acid)QS-18 Quillaic acid Gal-(1→2)-[Xyl-(1→3)]-GlcA-Api/Xyl-(1→3)-Xyl-(1→4)-[Glc-(1→3)]-Rha- (1→2)-[R-(→4)]-Fuc- (R =5-O-[5-O-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl- octanoic acid) QS-21 A-apioQuillaic acid Gal-(1→2)-[Xyl-(1→3)]-GlcA-Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R-(→4)]- Fuc- (R =5-O-[5-O-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl- octanoic acid) QS-21 A-xyloQuillaic acid Gal-(1→2)-[Xyl-(1→3)]-GlcA-Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R-(→4)]- Fuc- (R =5-O-[5-O-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl- octanoic acid) QS-21 B-apioQuillaic acid Gal-(1→2)-[Xyl-(1→3)]-GlcA-Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R-(→3)]- Fuc- (R =5-O-[5-O-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl- octanoic acid) QS-21 B-xyloQui!laic acid Gal-(1→2)-[Xyl-(1→3)]-GlcA-Xyl-(1→3)-Xyl-(1→4)-Rha-(1→2)-[R-(→3)]- Fuc- (R =5-O-[5-O-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl- octanoic acid) beta-AescinProtoaescigenin-21(2- Glc-(1→2)-[Glc-(1→4)]-GlcA- — (described:methylbut-2-enoate)-22- Aescin la) acetat Teaseed saponin I23-Oxo-barringtogenol C - Glc-(1→2)-Ara-(1→3)-[Gal- —21,22-bis(2-methylbut-2- (1→2)]-GlcA- enoate) Teaseedsaponin J23-Oxo-barringtogenol C - Xyl-(1→2)-Ara-(1→3)-[Gal- —21,22-bis(2-methylbut-2- (1→2)]-GlcA- enoate) Assamsaponin F23-Oxo-barringtogenol C - Glc-(1→2)-Ara-(1→3)-[Gal- —21(2-methylbut-2-enoate)- (1→2)]-GlcA- 16,22-diacetat DigitoninDigitogenin Glc-(1→3)-Gal-(1→2)-[Xyl- — (1→3)]-Glc-(1→4)-Gal- Primulaacid 1 3,16,28- Rha-(1→2)-Gal-(1→3)-[Glc- — Trihydroxyoleanan-12-en(1→2)]-GlcA- AS64R Gypsogenic acid — Glc-(1→3)-[Glc-(1→6)]-Gal-Carbohydrate substituent at the C-23-OH group AS6.2 Gypsogenic acid Gal-Glc-(1→3)-[Glc-(1→6)]-Gal- ^(a), ^(b)Different names refer to differentisolates of the same structure ^(c), ^(d)Different names refer todifferent isolates of the same structure

TABLE A2 ADCs which were previously investigated in the human clinicalsetting, and subsequently retracted from further clinical investigationLast Development Drug Name Indication Target Stage Monoclonal OncologyCells Expressing Epidermal Growth Factor Discovery Antibody Receptor(Proto Oncogene c ErbB 1 or Conjugate to Receptor Tyrosine ProteinKinase erbB 1 Target EGFR for or HER1 or ERBB1 or EGFR or EC Oncology2.7.10.1) Affilutin Multiple Myeloma (Kahler Disease) Discovery IMGN-779Myelodysplastic Syndrome Cells Expressing Myeloid Cell Surface IND/CTAFiled Antigen CD33 (Sialic Acid Binding Ig Like Lectin 3 or gp67 orCD33) Neuradiab Non-Hodgkin Lymphoma Cells Expressing Tenascin(Cytotactin or Phase I GMEM or GP 150-225 or Glioma AssociatedExtracellular Matrix Antigen or Hexabrachion or JI or MyotendinousAntigen or Neuronectin or Tenascin C or TNC) IMGN-779 Refractory AcuteMyeloid Leukemia; Cells Expressing Myeloid Cell Surface Phase I RelapsedAcute Myeloid Leukemia Antigen CD33 (Sialic Acid Binding Ig Like Lectin3 or gp67 or CD33) AGS-67E Acute Myelocytic Leukemia (AML, CellsExpressing Leukocyte Antigen CD37 Phase I Acute Myeloblastic Leukemia)(Tetraspanin 26 or CD37) AGS-67E Hairy Cell Leukemia; Non-Hodgkin CellsExpressing Leukocyte Antigen CD37 Phase I Lymphoma; Refractory Chronic(Tetraspanin 26 or CD37) Lymphocytic Leukemia (CLL); Relapsed ChronicLymphocytic Leukemia (CLL); T-Cell Leukemia ASG-15ME MetastaticTransitional (Urothelial) Cells Expressing SLIT And NTRK Like Phase ITract Cancer Protein 6 (SLITRK6) vandortuzumab Metastatic HormoneRefractory Cells Expressing Metalloreductase Phase I vedotin (CastrationResistant, Androgen- STEAP1 (Six Transmembrane Epithelial Independent)Prostate Cancer Antigen Of The Prostate 1 or STEAP1 or EC 1.16.1.)CDX-014 Ovarian Cancer Cells Expressing Hepatitis A Virus Cellular PhaseI Receptor 1 (Kidney Injury Molecule 1 or T Cell Immunoglobulin AndMucin Domain Containing Protein 1 or T-Cell Immunoglobulin MucinReceptor 1 or T Cell Membrane Protein 1 or CD365 or HAVCR1) AGS-16M18Liver Cancer; Renal Cell Carcinoma Phase I vorsetuzumab Non-HodgkinLymphoma; Renal Cell Cells Expressing CD70 Antigen (CD27 Phase Imafodotin Carcinoma Ligand or Tumor Necrosis Factor Ligand SuperfamilyMember 7 or CD70) denintuzumab Acute Lymphocytic Leukemia (ALL, CellsExpressing B Lymphocyte Antigen Phase I mafodotin Acute LymphoblasticLeukemia); B- CD19 (B Lymphocyte Surface Antigen B4 Cell Non-HodgkinLymphoma; Burkitt or Differentiation Antigen CD19 or T Cell Lymphoma;Lymphoblastic Surface Antigen Leu 12 or CD19) Lymphoma; Mantle CellLymphoma SGN-CD70A Diffuse Large B-Cell Lymphoma; Cells Expressing CD70Antigen (CD27 Phase I Follicular Lymphoma; Mantle Cell Ligand or TumorNecrosis Factor Ligand Lymphoma; Metastatic Renal Cell SuperfamilyMember 7 or CD70) Carcinoma; Non-Hodgkin Lymphoma RG-7636 MetastaticMelanoma Endothelin B Receptor (Endothelin Phase I Receptor NonSelective Type or EDNRB) SC-006 Metastatic Colorectal Cancer Phase IMM-310 Breast Cancer; Endometrial Cancer; Ephrin Type A Receptor 2(Epithelial Cell Phase I Esophageal Cancer; Gastric Cancer; Kinase orTyrosine Protein Kinase Gastroesophageal (GE) Junction Receptor ECK orEPHA2 or EC 2.7.10.1) Carcinomas; Head And Neck Cancer Squamous CellCarcinoma; Non-Small Cell Lung Cancer; Ovarian Cancer; Pancreatic DuctalAdenocarcinoma; Prostate Cancer; Small-Cell Lung Cancer; Soft TissueSarcoma; Solid Tumor; Transitional Cell Carcinoma (Urothelial CellCarcinoma) PF-06647263 Metastatic Breast Cancer; Ovarian CellsExpressing Ephrin A4 (EPH Related Phase I Cancer Receptor TyrosineKinase Ligand 4 or EFNA4) PF-06263507 Solid Tumor Cells ExpressingTrophoblast Glycoprotein Phase I (M6P1 or 5T4 Oncofetal Antigen or 5T4Oncofetal Trophoblast Glycoprotein or Wnt Activated Inhibitory Factor 1or TPBG) PF-06650808 Metastatic Breast Cancer; Non-Small CellsExpressing Neurogenic Locus Notch Phase I Cell Lung Cancer; OvarianCancer Homolog Protein 3 (NOTCH3) XMT-1522 Breast Cancer; GastricCancer; Non- Receptor Tyrosine Protein Kinase ERBB 2 Phase I Small CellLung Cancer (Metastatic Lymph Node Gene 19 Protein or Proto Oncogene Neuor Proto Oncogene C ErbB 2 or Tyrosine Kinase Type Cell Surface ReceptorHER2 or p185erbB2 or HER2 or CD340 or ERBB2 or EC 2.7.10.1); TubulinAMG-595 Anaplastic Astrocytoma; Recurrent Cells Expressing EpidermalGrowth Factor Phase I Glioblastoma Multiforme (GBM) Receptor (ProtoOncogene c ErbB 1 or Receptor Tyrosine Protein Kinase erbB 1 or HER1 orERBB1 or EGFR or EC 2.7.10.1) pinatuzumab Chronic Lymphocytic Leukemia(CLL) Cells Expressing B Cell Receptor CD22 (B Phase I vedotinLymphocyte Cell Adhesion Molecule or Sialic Acid Binding Ig Like Lectin2 or T Cell Surface Antigen Leu 14 or CD22) cantuzumab ColorectalCancer; Non-Small Cell Phase I ravtansine Lung Cancer; PancreaticCancer; Solid Tumor AVE-9633 Acute Myelocytic Leukemia (AML, CellsExpressing Myeloid Cell Surface Phase I Acute Myeloblastic Leukemia)Antigen CD33 (Sialic Acid Binding Ig Like Lectin 3 or gp67 or CD33)BIWI-1⁽¹⁾ Breast Cancer; Carcinomas; Cells Expressing CD44 Antigen(CDw44 or Phase I Esophageal Cancer; Head And Neck Epican orExtracellular Matrix Receptor III Cancer Squamous Cell Carcinoma or GP90Lymphocyte Homing/Adhesion Receptor or HUTCH I or Heparan SulfateProteoglycan or Hermes Antigen or Hyaluronate Receptor or PhagocyticGlycoprotein 1 or CD44) RG-7882 Epithelial Ovarian Cancer; FallopianCells Expressing Mucin 16 (Ovarian Phase I Tube Cancer; PancreaticCancer; Cancer Related Tumor Marker CA125 or Peritoneal Cancer OvarianCarcinoma Antigen CA125 or MUC16) ASG-5ME Adenocarcinoma; Hormone CellsExpressing Choline Transporter Like Phase I Refractory (CastrationResistant, Protein 4 (Solute Carrier Family 44 Androgen-Independent)Prostate Member 4 or SLC44A4) Cancer; Metastatic Adenocarcinoma of ThePancreas DCDS-0780A B-Cell Non-Hodgkin Lymphoma Phase I SC-004Endometrial Cancer; Epithelial Phase I Ovarian Cancer; Fallopian TubeCancer; Peritoneal Cancer RG-7600 Ovarian Cancer; Pancreatic DuctalPhase I Adenocarcinoma sofituzumab Epithelial Ovarian Cancer; FallopianCells Expressing Mucin 16 (Ovarian Phase I vedotin Tube Cancer; OvarianCancer; Cancer Related Tumor Marker CA125 or Pancreatic Cancer;Peritoneal Cancer Ovarian Carcinoma Antigen CA125 or MUC16) IMGN-289Breast Cancer; Esophageal Cancer; Cells Expressing Epidermal GrowthFactor Phase I Gastric Cancer; Head And Neck Receptor (Proto Oncogene cErbB 1 or Cancer Squamous Cell Carcinoma; Receptor Tyrosine ProteinKinase erbB 1 Non-Small Cell Lung Cancer; Solid or HER1 or ERBB1 or EGFRor EC Tumor 2.7.10.1) SAR-428926 Breast Cancer; Colorectal Cancer; CellsExpressing Lysosome Associated Phase I Gastric Cancer; Non-Small CellLung Membrane Glycoprotein 1 (CD107 Antigen Cancer; Ovarian Cancer;Prostate Like Family Member A or CD107a or Cancer; Solid Tumor LAMP1)SGNCD-19B B-Cell Non-Hodgkin Lymphoma; Cells Expressing B LymphocyteAntigen Phase I Diffuse Large B-Cell Lymphoma; CD19 (B LymphocyteSurface Antigen B4 Follicular Lymphoma or Differentiation Antigen CD19or T Cell Surface Antigen Leu 12 or CD19) SGNCD-123A Refractory AcuteMyeloid Leukemia; Cells Expressing Interleukin 3 Receptor Phase IRelapsed Acute Myeloid Leukemia Subunit Alpha (CD123 or IL3RA)SGNCD-352A Refractory Multiple Myeloma; Cells Expressing SLAM FamilyMember 6 Phase I Relapsed Multiple Myeloma (Activating NK Receptor or NKT B Antigen or CD352 or SLAMF6) RG-7841 Breast Cancer; Non-Small CellLung Cells Expressing Lymphocyte Antigen 6E Phase I Cancer; Solid Tumor(Retinoic Acid Induced Gene E Protein or Stem Cell Antigen 2 or ThymicShared Antigen 1 or LY6E) IMGN-388 Solid Tumor Cells Expressing IntegrinAlpha V Phase I (Vitronectin Receptor Subunit Alpha or CD51 or ITGAV)lorvotuzumab Refractory Multiple Myeloma; Cells Expressing Neural CellAdhesion Phase I mertansine Relapsed Multiple Myeloma Molecule 1(Antigen Recognized By Monoclonal Antibody 5.1H11 or CD56 or NCAM1)lorvotuzumab Neuroendocrine Carcinoma; Cells Expressing Neural CellAdhesion Phase I mertansine Neuroendocrine Tumors; Non-Small Molecule 1(Antigen Recognized By Cell Lung Cancer; Ovarian Cancer; MonoclonalAntibody 5.1H11 or CD56 or Skin Cancer NCAM1) BAY-794620 Lung Cancer;Solid Tumor Cells Expressing Carbonic Anhydrase 9 Phase I (CarbonateDehydratase IX or pMW1 or Membrane Antigen MN or P54/58N or Renal CellCarcinoma Associated Antigen G250 or CA9 or EC 4.2.1.1) RG-7598Refractory Multiple Myeloma; Phase I Relapsed Multiple Myeloma OncolysinB B-Cell Leukemia; Lymphoma Cells Expressing B Lymphocyte Antigen PhaseI CD19 (B Lymphocyte Surface Antigen B4 or Differentiation Antigen CD19or T Cell Surface Antigen Leu 12 or CD19) ADCT-502⁽¹⁾ Bladder Cancer;Breast Cancer; Cells Expressing Receptor Tyrosine Phase I EsophagealCancer; Gastric Cancer; Protein Kinase ERBB 2 (Metastatic LymphNon-Small Cell Lung Cancer Node Gene 19 Protein or Proto Oncogene Neu orProto Oncogene C ErbB 2 or Tyrosine Kinase Type Cell Surface ReceptorHER2 or p185erbB2 or HER2 or CD340 or ERBB2 or EC 2.7.10.1) AMG-172Renal Cell Carcinoma Cells Expressing CD70 Antigen (CD27 Phase I Ligandor Tumor Necrosis Factor Ligand Superfamily Member 7 or CD70)ImmuRAIT-LL2 B-Cell Non-Hodgkin Lymphoma Cells Expressing B CellReceptor CD22 (B Phase I/II Lymphocyte Cell Adhesion Molecule or SialicAcid Binding Ig Like Lectin 2 or T Cell Surface Antigen Leu 14 or CD22)indusatumab Adenocarcinoma Of The Gastro- Cells Expressing Heat StableEnterotoxin Phase I/II vedotin esophageal Junction; Gastric CancerReceptor (Guanylyl Cyclase C or or Intestinal Guanylate Cyclase orGUCY2C or EC 4.6.1.2) clivatuzumab Pancreatic Cancer Cells ExpressingMucin 1 (Breast Phase I/II tetraxetan Carcinoma Associated Antigen DF3or Episialin or H23AG or Krebs Von Den Lungen 6 or PEMT or PeanutReactive Urinary Mucin or Polymorphic Epithelial Mucin or TumorAssociated Epithelial Membrane Antigen or Tumor Associated Mucin orCD227 or MUC1) depatuxizumab Recurrent Malignant Glioma Epidermal GrowthFactor Receptor (Proto Phase I/II mafodotin⁽²⁾ Oncogene c ErbB 1 orReceptor Tyrosine Protein Kinase erbB 1 or HER1 or ERBB1 or EGFR or EC2.7.10.1) CDX-014 Metastatic Renal Cell Carcinoma; Cells ExpressingHepatitis A Virus Cellular Phase I/II Papillary Renal Cell CarcinomaReceptor 1 (Kidney Injury Molecule 1 or T Cell Immunoglobulin And MucinDomain Containing Protein 1 or T-Cell Immunoglobulin Mucin Receptor 1 orT Cell Membrane Protein 1 or CD365 or HAVCR1) vadastuximab RefractoryAcute Myeloid Leukemia; Cells Expressing Myeloid Cell Surface Phase I/IItalirine⁽¹⁾ Relapsed Acute Myeloid Leukemia Antigen CD33 (Sialic AcidBinding Ig Like Lectin 3 or gp67 or CD33) vadastuximab MyelodysplasticSyndrome Cells Expressing Myeloid Cell Surface Phase I/II talirineAntigen CD33 (Sialic Acid Binding Ig Like Lectin 3 or gp67 or CD33)MLN-2704 Metastatic Hormone Refractory Cells Expressing Glutamate PhaseI/II (Castration Resistant, Androgen- Carboxypeptidase 2 (FolateHydrolase 1 or Independent) Prostate Cancer Prostate Specific MembraneAntigen or PSMA or Pteroylpoly Gamma Glutamate Carboxypeptidase or CellGrowth Inhibiting Gene 27 Protein or FOLH1 or EC 3.4.17.21) Oncolysin BAIDS - Related Lymphoma Cells Expressing B Lymphocyte Antigen Phase I/IICD19 (B Lymphocyte Surface Antigen B4 or Differentiation Antigen CD19 orT Cell Surface Antigen Leu 12 or CD19) coltuximab Diffuse Large B-CellLymphoma Cells Expressing B Lymphocyte Antigen Phase II ravtansine CD19(B Lymphocyte Surface Antigen B4 or Differentiation Antigen CD19 or TCell Surface Antigen Leu 12 or CD19) coltuximab Acute LymphocyticLeukemia (ALL, Cells Expressing B Lymphocyte Antigen Phase II ravtansineAcute Lymphoblastic Leukemia) CD19 (B Lymphocyte Surface Antigen B4 orDifferentiation Antigen CD19 or T Cell Surface Antigen Leu 12 or CD19)coltuximab Diffuse Large B-Cell Lymphoma Cells Expressing B LymphocyteAntigen Phase II ravtansine CD19 (B Lymphocyte Surface Antigen B4 orDifferentiation Antigen CD19 or T Cell Surface Antigen Leu 12 or CD19)indusatumab Adenocarcinoma Of The Gastro- Cells Expressing Heat StableEnterotoxin Phase II vedotin⁽²⁾ esophageal Junction; Gastric Cancer;Receptor (Guanylyl Cyclase C or or Metastatic Adenocarcinoma of TheIntestinal Guanylate Cyclase or GUCY2C Pancreas or EC 4.6.1.2)depatuxizumab Squamous Non-Small Cell Lung Epidermal Growth FactorReceptor (Proto Phase II mafodotin Cancer Oncogene c ErbB 1 or ReceptorTyrosine Protein Kinase erbB 1 or HER1 or ERBB1 or EGFR or EC 2.7.10.1)depatuxizumab Anaplastic Astrocytoma; Anaplastic Epidermal Growth FactorReceptor (Proto Phase II mafodotin⁽²⁾ Oligoastrocytoma; Gliosarcoma;Oncogene c ErbB 1 or Receptor Tyrosine High-Grade Glioma; Oligoden-Protein Kinase erbB 1 or HER1 or ERBB1 droglioma; Pediatric DiffuseIntrinsic or EGFR or EC 2.7.10.1) Pontine Glioma; Recurrent GlioblastomaMultiforme (GBM) lifastuzumab Non-Small Cell Lung Cancer SodiumDependent Phosphate Transport Phase II vedotin Protein 2B (SodiumPhosphate Transport Protein 2B or NaPi3b or Sodium/PhosphateCotransporter 2B or NaPi 2b or Solute Carrier Family 34 Member 2 orSLC34A2) lifastuzumab Ovarian Cancer Sodium Dependent PhosphateTransport Phase II vedotin Protein 2B (Sodium Phosphate TransportProtein 2B or NaPi3b or Sodium/Phosphate Cotransporter 2B or NaPi 2b orSolute Carrier Family 34 Member 2 or SLC34A2) Bismab-A Acute MyelocyticLeukemia (AML, Cells Expressing Myeloid Cell Surface Phase II AcuteMyeloblastic Leukemia) Antigen CD33 (Sialic Acid Binding Ig Like Lectin3 or gp67 or CD33) denintuzumab Diffuse Large B-Cell Lymphoma; CellsExpressing B Lymphocyte Antigen Phase II mafodotin Follicular LymphomaCD19 (B Lymphocyte Surface Antigen B4 or Differentiation Antigen CD19 orT Cell Surface Antigen Leu 12 or CD19) Avicidin⁽¹⁾ Colorectal Cancer;Prostate Cancer Cells Expressing Epithelial Cell Adhesion Phase IIMolecule (Adenocarcinoma Associated Antigen or Cell Surface GlycoproteinTrop 1 or Epithelial Cell Surface Antigen or Epithelial Glycoprotein 314or KS 1/4 Antigen or KSA or Tumor Associated Calcium Signal Transducer 1or CD326 or EPCAM) pinatuzumab Diffuse Large B-Cell Lymphoma; CellsExpressing B Cell Receptor CD22 (B Phase II vedotin Follicular LymphomaLymphocyte Cell Adhesion Molecule or Sialic Acid Binding Ig Like Lectin2 or T Cell Surface Antigen Leu 14 or CD22) SGN-15 Metastatic BreastCancer; Non-Small Cells Expressing Lewis Y Antigen (CD174) Phase II CellLung Cancer; Ovarian Cancer; Prostate Cancer cantuzumab Gastric Cancer;Gastroesophageal Phase II ravtansine (GE) Junction Carcinomas ASP-6183Ovarian Cancer Phase II SAR-566658 Metastatic Breast Cancer CellsExpressing Sialoglycotope CA6 Phase II Antigen Oncolysin S Small-CellLung Cancer Cells Expressing Neural Cell Adhesion Phase II Molecule 1(Antigen Recognized By Monoclonal Antibody 5.1H11 or CD56 or NCAM1)lorvotuzumab Small-Cell Lung Cancer Cells Expressing Neural CellAdhesion Phase II mertansine Molecule 1 (Antigen Recognized ByMonoclonal Antibody 5.1H11 or CD56 or NCAM1) glembatumumab MetastaticMelanoma; Metastatic Cells Expressing Transmembrane Phase II vedotinUveal Melanoma; Osteosarcoma; Glycoprotein NMB (Transmembrane SquamousNon-Small Cell Lung Glycoprotein HGFIN or GPNMB) Cancer MM-302Metastatic Breast Cancer Cells Expressing Receptor Tyrosine Phase II/IIIProtein Kinase ERBB 2 (Metastatic Lymph Node Gene 19 Protein or ProtoOncogene Neu or Proto Oncogene C ErbB 2 or Tyrosine Kinase Type CellSurface Receptor HER2 or p185erbB2 or HER2 or CD340 or ERBB2 or EC2.7.10.1) Neuradiab Brain Cancer; Glioblastoma Cells Expressing Tenascin(Cytotactin or Phase III Multiforme (GBM) GMEM or GP 150-225 or GliomaAssociated Extracellular Matrix Antigen or Hexabrachion or JI orMyotendinous Antigen or Neuronectin or Tenascin C or TNC) clivatuzumabMetastatic Adenocarcinoma of The Cells Expressing Mucin 1 (Breast PhaseIII tetraxetan Pancreas Carcinoma Associated Antigen DF3 or Episialin orH23AG or Krebs Von Den Lungen 6 or PEMT or Peanut Reactive Urinary Mucinor Polymorphic Epithelial Mucin or Tumor Associated Epithelial MembraneAntigen or Tumor Associated Mucin or CD227 or MUC1) depatuxizumabGlioblastoma Multiforme (GBM) Epidermal Growth Factor Receptor (ProtoPhase III mafodotin⁽²⁾ Oncogene c ErbB 1 or Receptor Tyrosine ProteinKinase erbB 1 or HER1 or ERBB1 or EGFR or EC 2.7.10.1) vadastuximabAcute Myelocytic Leukemia (AML, Cells Expressing Myeloid Cell SurfacePhase III talirine⁽¹⁾ Acute Myeloblastic Leukemia) Antigen CD33 (SialicAcid Binding Ig Like Lectin 3 or gp67 or CD33) glembatumumab MetastaticBreast Cancer Cells Expressing Transmembrane Phase III vedotin⁽²⁾Glycoprotein NMB (Transmembrane Glycoprotein HGFIN or GPNMB) Oncolysin BB-Cell Leukemia; Lymphoma Cells Expressing B Lymphocyte Antigen PhaseIII CD19 (B Lymphocyte Surface Antigen B4 or Differentiation AntigenCD19 or T Cell Surface Antigen Leu 12 or CD19) ImmuRAIT-LL2 B-CellLeukemia Cells Expressing B Cell Receptor CD22 (B Preclinical LymphocyteCell Adhesion Molecule or Sialic Acid Binding Ig Like Lectin 2 or T CellSurface Antigen Leu 14 or CD22) indusatumab Metastatic Colorectal CancerCells Expressing Heat Stable Enterotoxin Preclinical vedotin Receptor(Guanylyl Cyclase C or or Intestinal Guanylate Cyclase or GUCY2C or EC4.6.1.2) ASG-15ME Lung Cancer Cells Expressing SLIT And NTRK LikePreclinical Protein 6 (SLITRK6) HTI-1511 Bile Duct Cancer CellsExpressing Epidermal Growth Factor Preclinical (Cholangiocarcinoma);Breast Cancer; Receptor (Proto Oncogene c ErbB 1 or Colorectal Cancer;Non-Small Cell Receptor Tyrosine Protein Kinase erbB 1 Lung Cancer orHER1 or ERBB1 or EGFR or EC 2.7.10.1) ZW-33 Gastric Cancer; MetastaticBreast Cells Expressing Receptor Tyrosine Preclinical Cancer ProteinKinase ERBB 2 (Metastatic Lymph Node Gene 19 Protein or Proto OncogeneNeu or Proto Oncogene C ErbB 2 or Tyrosine Kinase Type Cell SurfaceReceptor HER2 or p185erbB2 or HER2 or CD340 or ERBB2 or EC 2.7.10.1)ZW-33 Ovarian Cancer Cells Expressing Receptor Tyrosine PreclinicalProtein Kinase ERBB 2 (Metastatic Lymph Node Gene 19 Protein or ProtoOncogene Neu or Proto Oncogene C ErbB 2 or Tyrosine Kinase Type CellSurface Receptor HER2 or p185erbB2 or HER2 or CD340 or ERBB2 or EC2.7.10.1) SGNCD-352A Non-Hodgkin Lymphoma Cells Expressing SLAM FamilyMember 6 Preclinical (Activating NK Receptor or NK T B Antigen or CD352or SLAMF6) HuMax-CD74-ADC Oncology Cells Expressing HLA Class IIPreclinical Histocompatibility Antigen Gamma Chain (HLA DR AntigensAssociated Invariant Chain or Ia Antigen Associated Invariant Chain orp33 or CD74) sacituzumab Pancreatic Ductal Adenocarcinoma CellsExpressing Tumor Associated govitecan Calcium Signal Transducer 2 (CellSurface Glycoprotein Trop 2 or Membrane Component Chromosome 1 SurfaceMarker 1 or Pancreatic Carcinoma Marker Protein GA733-1 or TACSTD2)sacituzumab Adenocarcinoma; Cervical Cancer; Cells Expressing TumorAssociated govitecan Colorectal Cancer; Endometrial Calcium SignalTransducer 2 (Cell Surface Cancer; Epithelial Ovarian Cancer;Glycoprotein Trop 2 or Membrane Esophageal Cancer; Follicular ThyroidComponent Chromosome 1 Surface Cancer; Gastric Cancer; GlioblastomaMarker 1 or Pancreatic Carcinoma Marker Multiforme (GBM); Head And NeckProtein GA733-1 or TACSTD2) Cancer Squamous Cell Carcinoma;Hepatocellular Carcinoma; Kidney Cancer (Renal Cell Cancer); MetastaticHormone Refractory (Castration Resistant, Androgen- Independent)Prostate Cancer; Metastatic Transitional (Urothelial) Tract Cancer;Transitional Cell Cancer (Urothelial Cell Cancer) sacituzumabHepatocellular Carcinoma Cells Expressing Tumor Associated govitecanCalcium Signal Transducer 2 (Cell Surface Glycoprotein Trop 2 orMembrane Component Chromosome 1 Surface Marker 1 or Pancreatic CarcinomaMarker Protein GA733-1 or TACSTD2) sacituzumab Metastatic Breast Cancer;Transitional Cells Expressing Tumor Associated govitecan Cell Cancer(Urothelial Cell Cancer) Calcium Signal Transducer 2 (Cell SurfaceGlycoprotein Trop 2 or Membrane Component Chromosome 1 Surface Marker 1or Pancreatic Carcinoma Marker Protein GA733-1 or TACSTD2) sacituzumabNon-Small Cell Lung Cancer; Small- Cells Expressing Tumor Associatedgovitecan Cell Lung Cancer Calcium Signal Transducer 2 (Cell SurfaceGlycoprotein Trop 2 or Membrane Component Chromosome 1 Surface Marker 1or Pancreatic Carcinoma Marker Protein GA733-1 or TACSTD2) sacituzumabMetastatic Breast Cancer Cells Expressing Tumor Associated govitecanCalcium Signal Transducer 2 (Cell Surface Glycoprotein Trop 2 orMembrane Component Chromosome 1 Surface Marker 1 or Pancreatic CarcinomaMarker Protein GA733-1 or TACSTD2) ⁽¹⁾Discontinued due to adverse events⁽²⁾Discontinued due to lack of efficacy

TABLE A3 ADCs that reached phase III clinical development LastDevelopment Development Drug Name Indication Stage Stage Reason forDiscontinuation trastuzumab emtansine Gastric Cancer Marketed PhaseII/III Unspecified MM-302 Metastatic Breast Discontinued Phase II/IIIBusiness/Strategic Decision Cancer trastuzumab emtansine MetastaticBreast Marketed Phase III Unspecified Cancer trastuzumab emtansineGastric Cancer Marketed Phase III Unspecified ibritumomab tiuxetanDiffuse Large B- Marketed Phase III Cell Lymphoma inotuzumab ozogamicinFollicular Lymphoma Marketed Phase III inotuzumab ozogamicin DiffuseLarge B- Marketed Phase III Lack of Efficacy Cell Lymphoma; Non-HodgkinLymphoma rovalpituzumab tesirine Small-Cell Lung Phase III Phase IIICancer rovalpituzumab tesirine Small-Cell Lung Phase III Phase IIICancer Neuradiab Brain Cancer; Inactive Phase III UnspecifiedGlioblastoma Multiforme (GBM) clivatuzumab tetraxetan MetastaticInactive Phase III Unspecified Adenocarcinoma of The Pancreasdepatuxizumab mafodotin Glioblastoma Inactive Phase III Lack of EfficacyMultiforme (GBM) vadastuximab talirine Acute Myelocytic DiscontinuedPhase III Adverse Events Leukemia (AML, Acute Myeloblastic Leukemia)glembatumumab vedotin Metastatic Breast Discontinued Phase III Lack ofEfficacy Cancer Oncolysin B B-Cell Leukemia; Discontinued Phase IIIBusiness/Strategic Decision Lymphoma

TABLE A4 Tumor-specific cell-surface receptor targets which can betargeted by immunoglobulins according to the invention, and antibodiesthat can be used for the ADCs and the antibodies provided with asaponin, and the ADCs provided with a saponin, of the present invention(not presented as a limitation; further immunoglobulins are equallysuitable for the invention) Target cell- surface receptor Examplemonoclonal antibodies HER2 anti-HER2 monoclonal antibody such astrastuzumab and pertuzumab CD20 anti-CD20 monoclonal antibody such asrituximab, ofatumumab, tositumomab and ibritumomab CA125 anti-CA125monoclonal antibody such as oregovomab EpCAM (17-1A) anti-EpCAM (17-1A)monoclonal antibody such as edrecolomab EGFR anti-EGFR monoclonalantibody such as cetuximab, panitumumab and nimotuzumab CD30 anti-CD30monoclonal antibody such brentuximab CD33 anti-CD33 monoclonal antibodysuch as gemtuzumab and huMy9-6 vascular integrin anti-vascular integrinalpha-v beta-3 monoclonal antibody such as alpha-v beta-3 etaracizumabCD52 anti-CD52 monoclonal antibody such as alemtuzumab CD22 anti-CD22monoclonal antibody such as epratuzumab CEA anti-CEA monoclonal antibodysuch as labetuzumab CD44v6 anti-CD44v6 monoclonal antibody such asbivatuzumab FAP anti-FAP monoclonal antibody such as sibrotuzumab CD19anti-CD19 monoclonal antibody such as huB4 CanAg anti-CanAg monoclonalantibody such as huC242 CD56 anti-CD56 monoclonal antibody such huN901CD38 anti-CD38 monoclonal antibody such as daratumumab CA6 anti-CA6monoclonal antibody such as DS6 IGF-IR anti-IGF-IR monoclonal antibodysuch as cixutumumab and 3B7 integrin anti-integrin monoclonal antibodysuch as CNTO 95 syndecan-1 anti-syndecan-1 monoclonal antibody such asB-B4

TABLE A5 RIPs from plants* Plant Family Plant Species ProteinsClassification Adoxaceae Sambucus ebulus L. Ebulitin α, Ebulitin β,Ebulitin γ RIP 1 Ebulin f, Ebulin I, Ebulin r1, Ebulin r2, SEA RIP 2SEAII, SELfd, SELId, SELIm lectin Sambucus nigra L. α-Nigritin,β-Nigritin, γ-Nigritin, Nigritin f1, Nigritin f2 RIP 1 basic Nigrin b,Nigrin b = SNA-V, Nigrin f = SNA-Vf, RIP 2 Nigrin I1, Nigrin I2, Nigrins, SNA-I, SNA-I′, SNA-If, SNAflu-I, SNLRP1, SNLRP2 SNA-Id, SNA-Im,SNA-II, SNA-III, SNA-IV = SNA-IVf, lectin SNA-IVl, SNApol-I, SNApol-II,TrSNA-I, TrSNA-If Sambucus racemosa L. basic racemosin b, SRA RIP 2SRLbm = SRAbm lectin Sambucus sieboldiana SSA = SSA-b-1, Sieboldin-b =SSA-b-2 RIP 2 (Miq.) Blume ex Graebn. SSA-b-3, SSA-b-4 lectin AizoaceaeMesembryanthemum RIP1 RIP 1 crystallinum L. Amaranthaceae Amaranthuscaudatus L. Amaranthin = ACA lectin Amaranthus cruentus L. ACL lectinAmaranthus A. leucocarpus lectin lectin hypochondriacus L. [Syn.:Amaranthus leucocarpus S. Watson] Amaranthus mangostanus L. AmaramanginRIP 1 Amaranthus tricolor L. AAP-27 RIP 1 Amaranthus viridis L.Amaranthin RIP 1 Beta vulgaris L. Beetin-27 = BE27, Beetin-29 = BE29,Betavulgin RIP 1 Celosia argentea L. [Syn.: CCP-25, CCP-27 RIP 1 Celosiacristata L.] Chenopodium album L. CAP30 RIP 1 Spinacia oleracea L.SoRIP1 = BP31 RIP 1 SoRIP2 RIP 1 candidate Araliaceae Aralia elata(Miq.) Seem. Aralin RIP 2 Panax ginseng C. A. Mey Panaxagin peculiar RIP1 candidate/RNase Panax quinquefolius L. Quinqueginsin peculiar RIP 1candidate/RNase Asparagaceae Asparagus officinalis L. Asparin 1, Asparin2 RIP 1 Drimia maritima (L.) Stearn Charybdin RIP 1 [Syn.: Charybdismaritima (L.) Speta] Muscari armeniacum Musarmin 1, Musarmin 2, Musarmin3, Musarmin 4 RIP 1 Leichtlin ex Baker Polygonatum multiflorum PMRIPm,PMRIPt RIP 2 (L.) All. Yucca gloriosa var. tristis Yucca leaf protein =YLP RIP 1 Carrière [Syn.: Yucca recurvifolia Salisb.] BasellaceaeBasella rubra L. Basella RIP 2a, Basella RIP 2b, Basella RIP 3 RIP 1Caryophyllaceae Agrostemma githago L. Agrostin 2, Agrostin 5, Agrostin6, Agrostin RIP 1 Dianthus barbatus L. Dianthin 29 RIP 1 Dianthuscaryophyllus L. Dianthin 30, Dianthin 32 RIP 1 Dianthus chinensis L.[Syn.: D. sinensis RIP RIP 1 Dianthus sinensis Link] Gypsophila elegansM. Bieb. Gypsophilin RIP 1 Silene chalcedonica (L.) Lychnin RIP 1 E. H.L. Krause [Syn.: Lychnis chalcedonica L.] Silene glaucifolia Lag. [Syn.:Petroglaucin 1, Petroglaucin 2 RIP 1 Petrocoptis glaucifolia (Lag.)Boiss.] Silene laxipruinosa Mayol & Petrograndin RIP 1 Rosselló [Syn.:Petrocoptis grandiflora Rothm.] Saponaria ocymoides L. Ocymoidin RIP 1Saponaria officinalis L. Saporin-L1 = SO-L1, Saporin-L2 = SO-L2,Saporin-L3 = RIP 1 SO-L3, Saporin-I = SO-I = SO-4, Saporin-R1 = SO-R1,Saporin-R2 = SO-R2, Saporin-R3 = SO-R3, SO3a, SO3b, Saporin-S5 = Saporin5 = SO-S5, Saporin-S6 = Saporin 6 = SO-6 = SO-S6, Saporin-S8 = SO-S8,Saporin-S9 = Saporin 9 = SO-S9, SAP-C, SAP-S Myosoton aquaticum (L.)Stellarin RIP 1 Moench [Syn.: Stellaria aquatica (L.) Scop.] Stellariamedia (L.) Vill. RIP Q3 RIP 1 Vaccaria hispanica (Mill.) Pyramidatin RIP1 Rauschert [Syn.: Vaccaria pyramidata Medik.] Cucurbitaceae Benincasahispida (Thunb.) Hispin RIP 1 Cogn. α-benincasin, β-benincasin sRIP 1Bryonia cretica subsp. Bryodin 1 = BD1, Bryodin 2, Bryodin-L, Bryodin-RRIP 1 dioica (Jacq.) Tutin. [Syn.: BDA lectin/RIP 2 like Bryonia dioicaL.] Citrullus colocynthis (L.) Colocin 1, Colocin 2 RIP 1 Schrad.Cucurbita foetidissima Foetidissimin peculiar RIP 2 Kunth FoetidissiminII RIP 2 Cucumis ficifolius A. Rich. Cucumis figarei RIP = CF-RIP RIP 1candidate [Syn.: Cucumis figarei Delile ex Naudin] Cucurbita maximaCucurmoschin sRIP 1 candidate Duchesne Cucurbita moschata Cucurmosin,Cucurmosin 2, C. moschata RIP, RIP 1 Duchesne [Syn.: CucurbitaMoschatin, PRIP 1, PRIP 2 moschata (Duchesne ex α-moschin, β-moschinsRIP 1 candidate Lam.) Duchesne ex Poir.] Cucurbita pepo L. Pepocin RIP1 Cucurbita pepo var. texana Texanin RIP 1 (Scheele) D. S. Decker [Syn.:Cucurbita texana (Scheele) A. Gray] Gynostemma pentaphyllum GynostemminRIP 1 (Thunb.) Makino Lagenaria siceraria (Molina) Lagenin RIP 1candidate Standl. Luffa acutangula (L.) Roxb. Luffaculin-1, Luffaculin-2RIP 1 Luffangulin sRIP 1 Luffa acutangula fruit lectin lectin Luffacylindrica (L.) M. Roem Luffin, Luffin-a, Luffin-b, α-luffin, β-luffin,LRIP RIP 1 [Syn.: Luffa aegyptiaca Mill.] Luffacylin, Luffin P1 sRIP 1Luffin-S, LuffinS(1), LuffinS(2) = luffin S2, LuffinS(3) sRIP 1candidate Marah oreganus (Torr. & A. MOR-I, MOR-II RIP 1 Gray) HowellMomordica balsamina L. Balsamin, MbRIP-1, Momordin II RIP 1 Momordicacharantia L. MAP 30, α-momorcharin = α-MC = α-MMC, β- RIP 1 momorcharin= β-MC = β-MMC, δ-momorcharin = δ- MMC, Momordin, Momordin = Momordicacharantia inhibitor, Momordin II, Momordin-a, Momordin-b γ-momorcharin =γ-MMC, Charantin sRIP 1 RIP 1 candidate RIP 1 candidate MCL = M.charantia lectin, anti-H Lectin, Momordica lectin agglutinin, Momordin,protein fraction 1, protein fraction 2 MCL = Momordica charantia seedlectin = Momordica RIP 2 charantia lectin, MCL1 Momordicacochinchinensis Cochinin B, Momorcochin, Momorcochin-S RIP 1 Spreng.Siraitia grosvenorii (Swingle) Momorgrosvin RIP 1 C. Jeffrey ex A. M. Lu& Zhi Y. Zhang [Syn.: Momordica grosvenorii Swingle] Sechium edule(Jacq.) Sw. Sechiumin RIP 1 Sechium edule fruit lectin lectinTrichosanthes anguina L. Trichoanguin RIP 1 SGSL lectin/RIP 2 likeTrichosanthes cordata TCA-I, TCA-II lectin Roxb. Trichosanthescucumerina L. TCSL lectin/RIP 2 candidate Trichosanthes β-trichosanthin= β-TCS RIP 1 cucumeroides (Ser.) Maxim. Trichosanthes kirilowiiα-kirilowin, β-kirilowin, TAP 29, TK-35, Trichobitacin, RIP 1 Maxim.Trichokirin, Trichomislin = TCM, Trichosanthin = Trichosanthes antiviralprotein = TAP = TCS = α- trichosanthin = α-TCS = GLQ223, Trichosanthin,β- trichosanthin = β-TCS, γ-trichosanthin = γ-TCS Trichokirin S1,S-Trichokirin, Trichosanthrip sRIP 1 TKL-1 = Trichosanthes kirilowiilectin-1 lectin/RIP 2 candidate TK-I, TK-II, TK-III, Trichosantheskirilowii lectin lectin Trichosanthes kirilowii Karasurin-A,Karasurin-B, Karasurin-C RIP 1 Maximovicz var. japonica (Miquel)Kitamura Trichosanthes lepiniate Trichomaglin RIP 1 Trichosanthes dioicaRoxb. TDSL lectin/RIP 2 candidate Trichosanthes sp. Bac Kan TrichobakinRIP 1 8-98 Cupressaceae Thuja occidentalis L. Arborvitae RIP RIPcandidate Euphorbiaceae Croton tiglium L. Crotin I RIP 1 candidateCrotin 2 RIP 1 Euphorbia characias L. E. characias lectin lectinSuregada multiflora Gelonin = GAP 31 RIP 1 (A. Juss.) Baill. [Syn.:Gelonium multiflorum A. Juss.] Hura Crepitans L. Hura crepitans RIP,Hura crepitans RIP-5 RIP 1 Hura crepitans latex lectin RIP 2 Crepitin,Hurin, Hura crepitans seed lectin lectin Jatropha curcas L. Curcin,Curcin 2, Curcin-L, Jc-SCRIP RIP 1 Manihot palmata Müll. Arg. MapalminRIP 1 Manihot esculenta Crantz. Manutin 1, Manutin 2 RIP 1 [Syn.:Manihot utilissima Pohl] Ricinus communis L. Ricin = crystalline Ricin =Ricin D, Ricin E, RCA = RIP 2 Ricinus communis agglutinin = RCAI =RCA120 = R. communis hemagglutinin = RCB-PHA I, RCAII = RCA60 = RCB-PHAII Ricinus communis, USA Ricin 1, Ricin 2, Ricin 3 RIP 2 Ricinuscommunis, India Ricin I, Ricin II, Ricin III RIP 2 Ricinus sanguienus,France Ricin₁₁, Ricin₁₂, Ricin₂ RIP 2 Fabaceae Abrus precatorius L.Abrin, Abrin-a = Abrin C = Abrin-III, Abrin-b, Abrin-c = RIP 2 Abrin A =Abrin-I, Abrin-d, Abrin-II, APA = Abrus precatorius agglutinin = Abruslectin = AAG, APA-I, APA-II Abrus pulchellus Thwaites Pulchellin,Pulchellin PI, Pulchellin PII, Pulchellin PIII RIP 2 Pisum sativumsubsp. α-pisavin, β-pisavin RIP 1 sativum L. [Syn.: Pisum sativum var.arvense (L.) Poir.] Pisum sativum var. Sativin RIP 1 candidatemacrocarpon Iridaceae Iris hollandica var. Professor IrisRIP = IRIP,IrisRIP.A1, IrisRIP.A2, IrisRIP.A3 RIP 1 Blaauw IRA, IRAb, IRAr RIP 2Lamiaceae Clerodendrum aculeatum (L.) CA-SRI RIP 1 candidate Schltdl.Clerodendrum inerme (L.) CIP-29 RIP 1 Gaertn. CIP-34 RIP 1 candidateLeonurus japonicus Houtt. Leonurin RIP candidate Lauraceae Cinnamomumbodinieri H. Bodinierin RIP 2 Lév. Cinnamomum camphora (L.) CamphorinRIP 1 J. Presl Cinnamomin, Cinnamomin 1, Cinnamomin 2, RIP 2 Cinnamomin3 Cinphorin sRIP 2 Cinnamomum Porrectin RIP 2 parthenoxylon (Jack)Meisn. [Syn.: Cinnamomum porrectum (Roxb.) Kosterm.] MalvaceaeAbelmoschus esculentus Abelesculin RIP 1 (L.) Moench NyctaginaceaeBoerhaavia diffusa L. Boerhaavia inhibitor RIP 1 candidate Bougainvilleaspectabilis BAP I, Bouganin = Bougainvillea RIP I RIP 1 Willd.Bougainvillea × buttiana cv. BBP-24, BBP-28 RIP 1 Enid LancesterBougainvillea × buttiana cv. BBAP1 RIP 1 Mahara Mirabilis expansa (Ruiz& ME1, ME2 RIP 1 Pav.) Standl. Mirabilis jalapa L. MAP, MAP-2, MAP-3,MAP-4, MAP-S RIP 1 Olacaceae Malania oleifera Chun & Malanin lectin/RIP2 S. K. Lee candidate Ximenia americana L. Riproximin = Rpx, Rpx-I,Rpx-II RIP 2 Passifloraceae Adenia digitata (Harv.) Engl. Modeccin =Modeccin 4B, Modeccin 6B RIP 2 Adenia ellenbeckii Harms A. ellenbeckiilectin RIP 2 candidate Adenia fruticosa Burtt Davy A. fruticosa lectinlectin Adenia glauca Schinz A. glauca lectin RIP 2 candidate Adeniagoetzei Harms A. goetzei lectin RIP 2 (unresolved name) Adeniakeramanthus Harms A. keramanthus lectin RIP 2 candidate Adenialanceolata Engl. Lanceolin RIP 2 Adenia racemosa W. J. de A. racemosalectin lectin Wilde Adenia spinosa Burtt Davy A. spinosa lectin RIP 2candidate Adenia stenodactyla Harms Stenodactylin RIP 2 Adenia venenataForssk. A. venenata lectin RIP 2 candidate Adenia volkensii HarmsVolkensin RIP 2 Phytolaccaceae Phytolacca americana L. α-PAP, PAP =Phytolacca americana protein = RIP 1 pokeweed antiviral protein, PAP-I,PAP-II, PAP-III, PAP-C, PAP-H, PAP-R, PAP-S, PAP-S1, PAP-S2 Phytolaccadioica L. Diocin 1, Diocin 2, PD-L1, PD-L2, PD-L3, PD-L4, PD- RIP 1 S1,PD-S2, PD-S3 Phytolacca dodecandra Dodecandrin, Dodecandrin C RIP 1L'Hér. Phytolacca heterotepala Heterotepalin 4, Heterotepalin 5b RIP 1H. Walter Phytolacca insularis Nakai Insularin = PIP = Phytolaccainsularis antiviral protein, RIP 1 PIP2 = P. insularis antiviral protein2 Poaceae Hordeum vulgare L. Barley toxin = Barley translation inhibitor= Barley RIP 1 Protein Synthesis Inhibitor = BPSI = RIP 30, Barley toxinI = Barley translation inhibitor I, Barley toxin II = Barley translationinhibitor II = Barley Protein Synthesis Inhibitor II = BPSI II, Barleytoxin III = Barley translation inhibitor III, JIP60 Oryza sativa L.Oryza sativa RIP RIP 1 Secale cereale L. RPSI RIP 1 Triticum aestivum L.Tritin, Tritin 1, Tritin 2, Tritin 3, Tritin-S, Tritin-L RIP 1 Zea maysL. b-32 = maize RIP = maize proRIP1, Maize proRIP2 RIP 3/peculiar RIP 1Ranunculaceae Eranthis hyemalis (L.) EHL RIP 2 Salisb. SantalaceaePhoradendron californicum PCL RIP 2 Nutt. Viscum album L. HmRip, HmRip1, HmRip 2, HmRip 3, HmRip 4 RIP 2 (Himalayan mistletoe) Viscum album L.ML-I = Mistletoe lectin I = Viscumin = Eu-ML = EML-1 = RIP 2 (Europeanmistletoe) VAA-I, ML-II = Mistletoe lectin II = VAA-II, ML-III =Mistletoe lectin III = VAA-III Viscum articulatum Burm. f. Articulatin-DRIP 2 Viscum coloratum (Kom.) KML, KML-C, KML-IIL, KML-IIU, VCA RIP 2Nakai [Syn.: Viscum album subsp. coloratum Kom.] Solanaceae Nicotianatabacum L. CIP31 RIP-like protein TRIP RIP 1 candidate ThymelaeaceaePhaleria macrocarpa P. macrocarpa RIP RIP candidate (Scheff.) Boerl.*Schrot J, Weng A, Melzig M F, et al. Ribosome-inactivating and relatedproteins. Toxins (Basel). 2015 May 8; 7(5): 1556-615.

An aspect of the invention relates to a conjugate comprising orconsisting of an antibody and an antisense oligonucleotide such as anantisense BNA, covalently linked together. In FIG. 1-5, thegene-silencing activity of such a conjugate is depicted (in vivo test inan animal tumor model). Reference is also made to the Examples section.

An aspect of the invention relates to a combination of a firstcomposition comprising a conjugate comprising or consisting of anantibody and an antisense oligonucleotide such as an antisense BNA,covalently linked together, and a second composition comprising freesaponin of the invention (see Table A1, Scheme I). In FIG. 1-7A and inFIG. 1-7C, the gene-silencing activity of such a conjugate is depicted(in vitro cell-based bioassay with human tumor cells). Reference is alsomade to the Examples section.

An aspect of the invention relates to a pharmaceutical combinationcomprising or consisting of a first composition comprising a firstconjugate comprising or consisting of an antibody and an antisenseoligonucleotide such as an antisense BNA, and a second compositioncomprising a first conjugate comprising or consisting of the sameantibody and at least one saponin of the invention. In FIG. 1-5, thegene-silencing activity of such a conjugate is depicted (in vivo test inan animal tumor model). In FIG. 8-5, the gene-silencing activity of sucha conjugate is depicted (in vitro cell-based bioassay with human tumorcells). Reference is also made to the Examples section.

An aspect of the invention relates to a pharmaceutical combinationcomprising or consisting of a fourth composition comprising a fourthconjugate comprising or consisting of an antibody and an antisenseoligonucleotide such as an antisense BNA, and a fifth compositioncomprising a first conjugate comprising or consisting of a differentantibody and at least one saponin of the invention. In FIG. 10-6A and inFIG. 10-6C, the gene-silencing activity of such a conjugate is depicted(in vitro cell-based bioassay with human tumor cells). Reference is alsomade to the Examples section.

An aspect of the invention relates to a conjugate comprising orconsisting of an antisense oligonucleotide such as an antisense BNA,covalently linked to at least one saponin of the invention. In FIG. 1-3,the gene-silencing activity of such a conjugate is depicted (in vitrocell-based bioassay with human tumor cells). Reference is also made tothe Examples section.

An aspect of the invention relates to a conjugate comprising orconsisting of an antisense oligonucleotide such as an antisense BNA,covalently coupled to a polymeric scaffold such as a dendron such as aG4-dendron, wherein the polymeric scaffold is covalently conjugated withone or more saponin molecules of the invention, such as four saponinmolecules. In FIG. 1-3, the gene-silencing activity of such a conjugateis depicted (in vitro cell-based bioassay with human tumor cells).Reference is also made to the Examples section.

An aspect of the invention relates to a conjugate comprising orconsisting of an antibody such as a monoclonal antibody with specificityfor a tumor marker or tumor-cell receptor, covalently linked to at leastone antisense oligonucleotide molecule such as antisense BNA, andcovalently linked to at least one saponin molecule of the invention. InFIG. 2-4, the gene-silencing activity of such a conjugate is depicted(in vivo test in an animal tumor model). Reference is also made to theExamples section.

An aspect of the invention relates to a conjugate comprising orconsisting of an antibody such as a monoclonal antibody with specificityfor a tumor marker or tumor-cell receptor, covalently linked to at leastone antisense oligonucleotide molecule such as antisense BNA via atri-functional linker such as the linker of Scheme II, and covalentlylinked to at least one saponin molecule of the invention via the sametri-functional linker. In FIG. 1-1, the gene-silencing activity of sucha conjugate is depicted (in vivo test in an animal tumor model).Reference is also made to the Examples section.

An aspect of the invention relates to a therapeutic combinationconsisting or comprising of a eighth composition comprising a conjugatecomprising or consisting of an antibody, preferably a monoclonalantibody with specificity for a tumor marker or tumor-cell receptor,covalently linked to at least one saponin molecule of the invention,preferably via at least one linker, preferably at least one cleavablelinker, cleavable under physiological acidic conditions, and furthercomprising a ninth composition comprising an antisense oligonucleotidesuch as an antisense BNA molecule. In FIG. 6-2, the gene-silencingactivity of such a conjugate is depicted (in vivo test in an animaltumor model). In FIG. 5-2A and FIG. 5-2C, the gene-silencing activity ofsuch a conjugate is depicted (in vitro cell-based bioassay with humantumor cells). Reference is also made to the Examples section.

An aspect of the invention relates to any of the aforementionedconjugates or compositions or therapeutical combinations, for use as amedicament.

An aspect of the invention relates to any of the aforementionedconjugates or compositions or therapeutical combinations, for use in thetreatment or prophylaxis of a cancer.

Of course, as said before, any and all of a, b, c, d, e, f, g, h, I, j,k, m, n, p, q, r, s, t, u, v, w and/or x have the value in accordancewith each individual embodiment and aspect of the invention for any andall of the aforementioned aspects and embodiments according to theinvention. In addition, (tri-functional) linkers L1, L2, L4, L5, L6, L8,L9 and/or L10, if present in a molecule or conjugate or moiety of theinvention, are the (tri-functional) linkers as indicated for each andany of the aforementioned aspects and embodiments of the invention, asis readily appreciated by the skilled person. The oligomeric orpolymeric scaffolds L3 and/or L7, if present in a molecule or conjugateor moiety of the invention, are the oligomeric or polymeric scaffolds asindicated for each and any of the aforementioned aspects and embodimentsof the invention, as is also readily appreciated by the skilled person.Furthermore, the first ligand A1 and the first effector moiety B1, ifpresent, and the second ligand A2 and the second effector moiety B2, ifpresent, and the first effector moiety A1 and the first ligand B1, ifpresent, and the second effector moiety A2 and the second ligand B2, ifpresent, are the selected and indicated ligands and effector moieties,as disclosed for the first, second, third, fourth, fifth, and sixthseries of embodiment and aspects of the invention, and all furtherembodiments and aspects of the invention, outlined here above. Saponin Cis any one or more of the saponins referred to and listed in any of theaforementioned aspects and embodiments of the invention, in particularone or more saponins selected from Scheme I and/or Table A1.

The invention is further illustrated by the following examples, whichshould not be interpreted as limiting the present invention in any way.

EXAMPLES Example A—Treating a Mammalian Tumor-Bearing Animal with aConjugate of the Invention in Combination with an ADC Results inSurvival and Tumor Regression

Female Balb/c nude mice were injected subcutaneously with a suspensionof human A431 tumor cells. Under the skin of the mice, a human epidermalcarcinoma developed in the xenograft animal tumor model. After injectionof the tumor cells, the xenograft tumor was allowed to develop to a sizeof approximately 170-180 mm³. The A431 tumor cells have the followingcharacteristics: high EGFR expressors, medium CD71 expressors, low HER2expressors.

In Table A, the results of the treatment of control mice andtumor-bearing mice are presented. Tumor-bearing mice were treated withthe indicated antibodies directed to either human Her2/neu, human EGFR,or human CD71, which are cell-surface receptors on the xenograft tumor.Cetuximab was covalently conjugated with saponin SO1861. The SO1861 wasfirst provided with the linker EMCH (N-ε-maleimidocaproic acidhydrazide), which EMCH is a maleimide-and-hydrazide crosslinker forcovalently conjugating sulfhydryls (reduced cysteines of the antibody))to carbonyls (aldehyde or ketones; here the carbonyl of the aldehyde atposition C-23 of the saponin). The saponin-EMCH was covalently coupledto reduced cysteines of the Cetuximab, forming a covalent thio-etherbond between the EMCH and the cysteine side chain. The ADCstrastuzumab-saporin (covalent conjugate) and anti-CD71 mAb (OKT-9,IgG)—saporin (covalent conjugate) were tested for their tumor-attackingefficacy in the mice, measured as tumor volume in time after start ofthe treatment with the ADCs. The dose of the ADCs was sub-optimal in thetumor model. That is to say, from previous experiments, it wasestablished at which sub-optimal dose of the ADCs no tumor-regression orarrest of tumor growth would be observable.

TABLE A RESULTS OF TREATING A MAMMALIAN TUMOR-BEARING ANIMAL WITH ACONJUGATE OF THE INVENTION IN COMBINATION WITH AN ADC RESULTS INSURVIVAL AND TUMOR REGRESSION tumor size (volume in mm³ or ‘+’ forgrowth, ‘−’ for regression, Treatment Patient/healthy and ‘stable’ forgrowth nor group animal treatment regression) 1 xenograft vehicle 2000mm³ (death/euthanasia) 2 xenograft Trastuzumab-saporin 2000 mm³(death/euthanasia) 3 xenograft Anti-CD71 mAb OKT-9 − 2000 mm³(death/euthanasia) saporin (covalent conjugate) 4 xenograftCetuximab-SO1861 2000 mm³ (death/euthanasia) (covalent conjugate) 5xenograft Cetuximab >170 mm³, but <2000 mm³ (death/euthanasia) 6xenograft Trastuzumab-saporin Tumor regression from 180 mm³ (covalentconjugate) + at the start of treatment back to Cetuximab-SO1861 80 mm³(survival) (covalent conjugate) 7 xenograft Anti-CD71 mAb OKT-9 − Tumorregression from 180 mm³ saporin (covalent at the start of treatment backto conjugate) + Cetuximab- 40 mm³ (survival) SO1861 (covalent conjugate)

These results demonstrate that the combination therapy of an ADC at adose which is ineffective when treatment of tumor-bearing mice with theADC alone is considered (tumor growths, death of the mice is notprevented (euthanasia)), with a conjugate of the invention consisting ofa tumor-cell specific receptor targeting antibody covalently bound to asaponin, i.e. SO1861, the covalent conjugate administered to the micesuffering from cancer, at a non-effective dose when administered alone(tumor growths, death of the mice is not prevented (euthanasia)),provides an efficient and efficacious treatment regimen, expressed astumors in regression and prolonged survival of the treated animals(beyond the duration of the experiment). The sub-optimal dose of ADCcombined with a covalently bound saponin-comprising conjugate of theinvention which has no anti-tumor activity when administered alone, thusprovide for an effective treatment option for cancer patients, wherein arelative low dose of the ADC is efficacious. A lower dose of ADC bearsthe promise of less risk for adverse events, or even no side effects atall. In addition, the stimulatory effect of the saponin-bearingconjugate of the invention when the efficacy of the ADC is considered,shows that ADCs which previously have proven to lack efficacy when tumorpatient treatment is concerned, may gain renewed attention and value,since ADC efficacy is improved in combination therapy setting, as thecurrent example demonstrated. Reference is made to Table A2 and TableA3, summarizing ADCs which were previously investigated in the humanclinical setting, but then were for some ADCs retracted from furtherclinical investigation. Especially the ADCs for which clinicaldevelopment was terminated due to observed lack of efficacy and/or dueto occurrence of unacceptable adverse event are ADCs which may gainrenewed value for cancer patients when combined with a covalently boundsaponin-comprising conjugate of the invention, such as thecetuximab-saponin tested.

Example B—Saponins Mixture of Quillaja saponaria Comprising QS-21, withEndosomal/Lysosomal Escape Enhancing Activity

Scheme I displays the common molecular structure of a series of QS-21saponins (in part adapted from: Conrado Pedebos, Laércio Pol-Fachin,Ramon Pons, Cilaine V. Teixeira Hugo Verli, Atomic Model and MicelleDynamics of QS-21 Saponin, Molecules 2014, 19, 3744-3760). A mixture ofwater-soluble saponins obtained from Quillaja saponaria (Sigma-Aldrich,product No. S4521; Roth, Item No. 6857; InvivoGen, product ‘Quit-A’) maybe applied in the endosomal/lysosomal escape enhancing conjugate,composition, combination of the invention, based on endosomal/lysosomalescape enhancing properties of at least one individual saponin presentin the mixture, e.g. QS-21, or based on a combination of two or more ofthe saponins comprised by the mixture, such as QS-21 and QS-7.

The inventors demonstrated that the mixture of saponins from Quillajasaponaria at 2.5 microgram/ml dose was capable of enhancing endosomalescape of dianthin, as tested with mammalian tumor cells in a cell-basedbioassay. The effector moiety exposed to the cells was dianthincovalently coupled to the ligand EGF: EGF-dianthin. Cells tested weretumor cell lines HeLa for free saponins, and A431, MDA-MB-468, CaSki andA2058 for testing the saponins when covalently coupled to cetuximab.

Example 1

A trifunctional linker scaffold was designed and produced with specificchemical end groups (DBCO, TCO) for conjugation (labile, (L)conjugation) with on one arm an SO1861 molecule and on the other arm anantisense HSP27BNA oligo nucleotide (targeting and inducing degradationof the onco-target hsp27 mRNA in cancer cells) to produceSO1861-L-trifunctional linker-L-HSP27BNA (FIG. 16-1).SO1861-L-trifunctional linker-L-HSP27BNA was conjugated with its thethird arm (maleimide) to the cysteine residues (Cys) anti-EGFR antibody,cetuximab (cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA)⁴).

This scaffold comprising conjugate was tested in a A431 xenograph ‘nude’mouse tumor model for EGFR-mediated tumor targeted gene silencingactivity. Dosings started at day 12 when tumors reached ˜170 mm³ in sizeand tumor samples were collected at 72h after the first dosing andanalysed for HSP27 gene expression compared to cellular control mRNAexpression (reference genes). This revealed that 1 dosing of 25 mg/kgcetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA)³′⁷ resulted ina 40% reduction in HSP27 gene expression in the tumors compared tosingle dosing of cetuximab-(Cys-L-SO1861)^(3,8) orcetuximab-(Lys-L-HSP27BNA)⁴ mono therapies (FIG. 1-1). Compared to thevehicle control tumors a reduction of 25% gene silencing was observed.This shows and enables that conjugated SO1861 efficiently can inducetargeted delivery of therapeutic oligo nucleotides in tumors, in vivo.

To further strengthen this, cetuximab-Cys-(SO1861-L-trifunctionallinker-L-HSP27BNA DAR4)⁴ was tested for enhanced HSP27 gene silencing inEGFR expressing (A431), in vitro as illustrated in FIG. 2-1.Cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA)³′⁷ efficientlyinduces HSP27 gene silencing in A431 cells (IC50= . . . ) compared toCetuximab-(Lys-L-HSP27BNA)⁴ or Cetuximab-(Cys-L-SO1861)^(3,8) alone(FIG. 2-1).

Example 2

1 target 2-components system is the combination treatment ofmAb1-(dendron(SO1861)n)n and mAb1-effector as illustrated in FIG. 11-1and whereas the 2 target 2-component system is the combination ofmAb1-(dendron(SO1861)^(n))^(n) mAb2-effector as illustrated in FIG.12-1.

Dendron(-L-SO1861)⁴ was conjugated to the anti-EGFR antibody, cetuximabvia cysteine residues (Cys) conjugation with a DAR3,9,cetuximab-Cys-(dendron(-L-SO1861)⁴)^(3,9) and tested for enhanced cellkilling activity in combination with an anti-EGFR antibody-protein toxinconjugate (cetuximab-saporin) in EGFR expressing cells (MDA-MB-468).Cetuximab-Cys-(dendron(-L-SO1861)⁴)^(3,9)+10 pM cetuximab-saporinefficiently induces toxin-mediated cell killing in high EGFR expressingcells (IC50= . . . ), whereas this was not induced byCetuximab-Cys-(dendron(-L-SO1861)⁴)^(3,9) or cetuximab (equivalent)+10pM cetuximab-saporin or cetuximab (FIG. 3-1A). Similar experiments incells that express low levels of EGFR (HeLa) revealed no activity ofCetuximab-Cys-(dendron(-L-SO1861)⁴)^(3,9) (FIG. 3-1C) indicating that inthe absence of sufficient EGFR receptor expression, effectiveintracellular SO1861 concentrations are not reached (threshold) toinduce endosomal protein toxin escape and toxin-mediated cell killing.

Next, dendron(-L-SO1861)⁴ was conjugated to the anti-HER2 antibody,trastuzumab via cysteine conjugation (Cys) with a DAR4,trastuzumab-Cys-(dendron(-L-SO1861)⁴)⁴ and tested for enhanced cellkilling activity in combination with an anti-HER2 antibody-protein toxinconjugate (trastuzumab-saporin) in HER2 expressing cells (SK-BR-3).trastuzumab-Cys-(dendron(-L-SO1861)⁴)⁴+50 pM trastuzumab-saporinefficiently induce toxin-mediated cell killing (IC50= . . . ), whereasthis was not induced by trastuzumab-Cys-(dendron(-L-SO1861)⁴)⁴ ortrastuzumab (equivalent)+50 nM trastuzumab-saporin or trastuzumab (FIG.3-1B). Similar experiments in cells that express low levels of HER2(JIMT-1) revealed no activity of Trastuzumab-Cys-(dendron(-L-SO1861)⁴)⁴(FIG. 3-1D) indicating that in the absence of sufficient HER2 receptorexpression, effective intracellular SO1861 concentrations are notreached (threshold) to induce endosomal protein toxin escape andtoxin-mediated cell killing.

Next, Cetuximab-Cys-(dendron(-L-SO1861)⁴)^(3,9) orCetuximab-Lys-(dendron(-L-SO1861)⁴)^(4,4) (Lys=dendron(-L-SO1861)⁴conjugated to lysines of antibody) was tested in combination with 10 pMCD71mab-saporin in a 2 target 2 components system in EGFR⁺⁺/CD71⁺ cells(MDA-MB-468). This showed for both conjugates a strong enhancement ofthe cell killing activity (IC50= . . . IC50= . . . resp.), whereas thiswas not induced by Cetuximab-Cys-(dendron(-L-SO1861)⁴)^(3,9) orCetuximab-Lys-(dendron(-L-SO1861)⁴)^(4,4) or cetuximab (equivalent)+10pM CD71mab-saporin or cetuximab (FIG. 4-1A). Similar experiments incells that express lower levels of EGFR (CaSKi, EGFR⁺/CD71⁺) revealedreduced activity for both cetuximab-Cys-(dendron(-L-SO1861)⁴)^(3,9) orcetuximab-Lys-(dendron(-L-SO1861)⁴)^(4,4) (FIG. 4-1C) compared to theactivity in high expressors (FIG. 4-1A) indicating that in cells withlower EGFR receptor expression levels, the effective intracellularSO1861 concentrations is lower resulting in reduced toxin-mediated cellkilling activity.

Same experiment was performed withtrastuzumab-Cys-(dendron(-L-SO1861)⁴)⁴ ortrastuzumab-Lys-(dendron(-L-SO1861)⁴)⁴⁷ in combination withCD71mab-saporin on HER2⁺⁺/CD71+(SK-BR-3) cell lines revealing strongcell killing activity compared to the controls (FIG. 4-16). Whentrastuzumab-Cys-(dendron(-L-SO1861)⁴)⁴ ortrastuzumab-Lys-(dendron(-L-SO1861)⁴)^(4,7) was tested onHER2^(+/−)/CD71⁺ (JIMT-1) in combination with 10 pM CD71mab-saporin nocell killing activity could be observed indicating that in the absenceof sufficient HER2 receptor expression, effective intracellular SO1861concentrations are not reached (threshold) to induce endosomal proteintoxin escape and toxin-mediated cell killing.

Next, trastuzumab-Cys-(dendron(-L-SO1861)⁴)⁴+trastuzumab-emtansine(T-DM1, antibody-small molecule toxin conjugate) was tested for enhancedcell killing activity in HER2 expressing cells (SK-BR-3). No enhancedcell killing was observed with this combination, compared to T-DM1 aloneor T-DM1+equivalent trastuzumab, since the endosomal membrane forms nobarrier for small molecules to reach the cytoplasm. (FIG. 5-1).

Example 3 Materials and Methods Dendron(SO1861)₄-BNA Oligo Synthesis(FIG. 17-1)

HSP27BNA oligo disulfide (1.1 mg, 0.187 μmol) was dissolved in 20 mMNH₄HCO₃ with 1.0 mM TCEP (500 μL) and the mixture was shaken for 1 minand left standing at room temperature. After 1 hour the reaction mixturewas filtered by using a centrifugal filter with a molecular weightcut-off of 3000 Da (14000×g for 30 min). The residue solution wasdiluted with 20 mM NH₄HCO₃ with 1.0 mM TCEP (500 μL) and the resultingmixture was filtered again under the same conditions described above.The residue solution was diluted with 20 mM NH₄HCO₃/acetonitrile (3:1,v/v, 1.0 mL) and the resulting mixture was added todendron(SO1861)4-maleimide1 (3.54 mg, 0.375 μmol) (FIG. 17-1). Thereaction mixture was shaken for 1 min and left standing at roomtemperature. After 10 min the reaction mixture was subjected topreparative LC-MS.^(4A) Fractions corresponding to the product wereimmediately pooled together, frozen and lyophilized overnight to givethe title compound (1.25 mg, 85%) as a white fluffy solid. Purity basedon LC-MS 94%

LRMS (m/z): 1896 [M-8]⁸⁻, 2167 [M-7]⁷⁻

LC-MS r.t. (min): 3.77⁶⁸

Results

HSP27BNA oligo, (antisense BNA oligo targeting the mRNA transcript ofthe cancer target, heat shock protein 27 (HSP27BNA)) was conjugated to adendron(-L-SO1861)⁴ (HSP27BNA-dendron(-L-SO1861)⁴, FIG. 17-1) andco-administrated to A431 cancer cells. As readout, gene silencing ofHSP27 mRNA in A431 cells was determined. This revealed thatHSP27BNA-dendron(-L-SO1861)⁴ treatment resulted in an improvement ofHSP27 gene silencing activity compared to the HSP27BNA alone (FIG. 6-1).

Example 4 Methods SO1861 Releasing Assay

To dendron(SO1861)₄-Cbz (0.05 mg) (FIG. 7-1) was added 50 μL of solutioncontaining water/acetonitrile/TFA (1.00 mL/1.00 mL/4 drops). Thereaction mixture was shaken for 1 min and left standing at roomtemperature. The SO1861 release was followed overtime by using UPLC-MS.⁴

Results

The release efficiency of the SO1861 molecules from thedendron(-L-SO1861)⁴ under acid conditions has been determined (FIG.7-1).

Next, dendron(-L-SO1861)⁴ was tested for enhanced delivery of a targetedtoxin, EGFdianthin on EGFR expressing cells (A431 and HeLa). This showsthat dendron(L-SO1861)⁴+10 pM EGFdianthin can induce enhancedtoxin-mediated cell killing (IC50 . . . nM), whereas the ‘naked’ dendron(Dendron(NEM)⁴) or dendron(-L-SO1861)⁴ or Dendron(NEM)⁴+10 pMEGFdianthin is not showing enhanced cell killing at these concentrations(FIG. 8-1A, 8-1B).

Example 5 Materials and Methods

In our current work, we investigated a model scaffold consisting of fourmolecular arms for saponin binding via a Schiff base (imine) and one armfor click chemistry. The polymeric structure (FIG. 19-1) is apentavalent polyethylene glycol-based dendrimer of the first generation(i.e. number of repeated branching cycles) that was purchased from IrisBiotech GmbH (Marktredwitz, Germany). The saponin (in this exampleSA1641) was purified from a saponin composite raw extract fromGypsophila species called Saponinum album obtained from Merck(Darmstadt, Germany). The powdered raw extract (2.5 g) was hydrolyzed inwater (100 mL) with sodium hydroxide (0.2 g). The solution was stirredfor 20 h at 40° C. and then supplemented with glacial acetic acid untilpH 5.0 was reached. To remove tannins, the solution was shaken in aseparatory funnel with 30 mL butanol. The aqueous phase was recapturedand butanol extraction repeated two times. The butanol phases weresupplemented with anhydrous sodium sulfate, filtered and pooled. Butanolwas evaporated and the remaining saponin powder resolved in 20% methanolto a final concentration of 30 mg/mL. After short sonication, differentsaponins were separated by high performance liquid chromatography(HPLC). Tubes (excluding column) were rinsed with warm water (40° C.) ata flow of 1.5 mL/min and then including Eurospher RP-C18-column (5 μm,250×8 mm) with isopropanol (100%). Saponins were applied to the columnand eluted with a methanol gradient (20% methanol to 70% methanol within30 min at 1.5 mL/min in water supplemented with 0.01% trifluoroaceticacid followed by 70% methanol for further 60 min) (Sama et al, 2018).Aliquots of the fractions were analyzed for their SA1641 content byelectrospray ionization mass spectrometry (ESI-MS). Fractions containingpure SA1641 were pooled and methanol evaporated. The aqueous solutionwas frozen as a thin film in a rotating round-bottom flask by use of dryice. After storage for 16 h at −80° C., the sample was lyophilized. Toproduce the scaffold as defined in the invention, the polymericstructure (0.2 mM) and SA1641 (3.2 mM) were solved in water (approx. pH8) and equal volumes mixed and shaken for 24 h at 26° C. Then sodiumcyanoborohydride (NaCNBH₃; 0,1 M) was added in 4-fold molar excessreferred to SA1641 and the sample incubated for further 24 h. Thestructure was then verified by ultra performance liquid chromatography(UPLC)/ESI-MS. The samples were applied to a RP-C4-column and elutedwith a methanol gradient (25% methanol to 80% methanol within 15 min inwater supplemented with 0.01% trifluoroacetic acid followed by 80%methanol for further 10 min). The fractions were analyzed by use ofLockSpray™ that is an ion source designed specifically for exact massmeasurement with electrospray ionization using LC-time-of-flight(LC-TOF) mass spectrometers from Waters Corporation.

Example 6 Materials and Methods

As an example for a pharmaceutical active substance, we used thetargeted toxin dianthin-Epidermal Growth Factor (dianthin-EGF). Theplasmid His-dianthin-EGF-pET11d (Weng et al, 2009) (100 ng) was added to20 μL Escherichia coli Rosetta™ 2 (DE3) pLysS Competent Cells (Novagen,San Diego, Calif., USA). Cells were transformed by a heat-shock (30 minon ice, 90 s at 42° C. and 1 min on ice). Thereafter, 300 μL lysogenybroth (LB) was added and the suspension incubated for 1 h at 37° C.while shaking at 200 rpm. A preheated lysogeny broth agar plate with 50pg/mL ampicillin was inoculated with 100 μl bacteria suspension and theplate incubated overnight at 37° C. Lysogeny broth (3 mL) with 50 pg/mLampicillin was inoculated with a colony from the plate and the bacteriawere incubated for 8 h at 37° C. and 200 rpm. The suspension (50 μL) wasadded to 500 mL of lysogeny broth with 50 pg/mL ampicillin and incubatedovernight at 37° C. and 200 rpm. Subsequently, the volume was scaled-upto 2.0 L and bacteria grew under the same conditions until an opticaldensity at wavelength 600 nm of 0.9 was reached. Thereafter, proteinexpression was induced by the addition of isopropylβ-D-1-thiogalactopyranoside (IPTG) at a final concentration of 1 mM.Protein expression lasted for 3 h at 37° C. and 200 rpm. Finally, thebacterial suspension was centrifuged at 5,000×g and 4° C. for 5 min,resuspended in 20 mL PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 1.47mM KH₂PO₄) and stored at −20° C. until use. For purification, bacterialsuspensions were thawed and lysed by sonication. Lysates werecentrifuged (15,800×g, 4° C., 30 min) and imidazole added to a finalconcentration of 20 mM. The supernatant was incubated with 2 mL ofNi-nitrilotriacetic acid agarose under continuous shaking for 30 min at4° C. in the presence of 20 mM imidazole. Subsequently, the material waspoured into a 20-mL-column and washed three times with 10 mL wash buffer(50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole) and dianthin-EGF eluted by10-mL-portions of increasing concentrations of imidazole (31, 65, 125and 250 mM) in wash buffer. Eluate fractions (2 mL) were dialyzedovernight at 4° C. against 2.0 L PBS. Desalted dianthin-EGF wasconcentrated by an Amicon® Ultra-15 (10 kDa) and the proteinconcentration quantified.

To introduce a suitable click chemistry group into dianthin-EGF,alkyne-PEG₅-N-hydroxysuccinimidyl ester in 8-fold molar excess referredto dianthin-EGF was solved in dimethyl sulfoxide and added to 9 volumesof dianthin-EGF (1 mg in 0.2 M NaH₂PO₄/Na₂HPO₄, pH 8). After incubationat room temperature for 4 h, non-bound alkyne was separated by use of aPD10 column (GE-Healthcare, Freiburg, Germany). Click chemistry with thepolymeric structure was conducted by copper(I)-catalyzed alkyne-azidecycloaddition. Alkyne-dianthin-EGF (0.02 mM), dendrimer (0.05 mM), CuSO4(0.1 mM), tris(3-hydroxypropyltriazolylmethyl)amine (0.5 mM) and sodiumascorbate (5 mM) were incubated under gentle agitation for 1 h at roomtemperature in 0.1 M NaH₂PO₄/Na₂HPO₄, pH 8. Low molecular masssubstances were then separated using a PD10 column.

To test the efficacy of the invention, we conducted a viability assaywith HER14 cells. These cells are fibroblasts stably transfected withthe human epidermal growth factor receptor and therefore target cellsfor the targeted toxin dianthin-EGF. HER14 cells (2,000 cells/100μL/well) were seeded into wells of 96-well-cell culture plates andincubated for 24 h in DMEM medium supplemented with 10% fetal calf serumand 1% penicillin/streptomycin at 37° C., 5% CO₂ and 98% humidity. Thedifferent test substances (see results and FIG. 21-1) were then added intriplicates in a volume of 25 μL and supplemented with further 25 μL ofmedium. After an incubation of 72 h, 30 μL3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (0.5 mg/mLin water) was added per well and incubated for 2 h. Thereafter, themedium was carefully removed and replaced by an aqueous solutioncontaining 10% (v/v) isopropanol, 5% (w/v) sodium dodecyl sulfate and400 mM HCl, and incubated for 5 min. Solubilized formazan wasphotometrically quantitated at 570 nM in a microplate reader (SpectraMAX 340 PC, Molecular Devices, Sunnyvale, Calif., USA). Untreated cellswere normalized to 1 and all samples referred to the untreated control.Significance was determined by unpaired two-sample t-tests.

Results

The polymeric structure, in the example a pentameric dendrimer(pentrimer), does not have any cytotoxic effect on the target cells,neither in absence nor in presence of SA1641 (FIG. 21-1, column 2 and3). In the absence of the scaffold, the targeted toxin (dianthin-EGF)shows half maximal toxicity at a concentration of 0.1 nM (column 4). Inthe presence of SA1641 the same concentration results in death of allcells indicating the general ability of SA1641 to act as an enhancer ofthe endosomal escape (column 5). The presence of the polymeric structuredoes not affect the toxicity of dianthin-EGF neither in the presence norin the absence of SA1641 (columns 6 and 7), indicating that the scaffolddoes not affect the toxicity of dianthin-EGF. To couple the modelpolymeric structure via click chemistry to the example pharmaceuticallyactive substance of dianthin-EGF, the substance had to be coupled withan alkyne group before. A manufacturer of a pharmaceutically activesubstance can introduce the click position during synthesis directlyinto the substance at a position of his choice where the activity of thesubstance remains unaffected. There was no additional loss of activitywhen clicking the alkyne-modified pharmaceutically active substance tothe polymeric structure indicating that the polymeric structure itselfwas not toxic.

Example 7

Considering available chemical groups for conjugation reactions to theSO1861 molecule, four chemical groups have been identified. The alcoholsand diols of the sugar residues, the aldehyde group on the triterpenoidbackbone, the carboxylic acid on one of the sugar residues (glucuronicacid), and the alkene group on the triterpenoid backbone as highlightedin FIG. 19-1.

In view of the pros and cons of each identified chemical group (Table1), the aldehyde and alcohol groups are best suitable for reversibleconjugation reactions, while the alkene and the carboxylic acid(glucuronic acid) are the groups best suitable for irreversible/stableconjugation reactions. The aldehyde group within the molecule structureof SO1861, however, is the most suitable for reversible conjugationreactions over the alcohols. On the one hand, because there is only onealdehyde present in the structure that allows chemoselective reactions.On the other hand, because the aldehyde can perform reversibleconjugation reactions with a variety of chemical groups such as amines,hydrazides, and hydroxylamines forming acid-cleavable moieties likeimines, hydrazones, and oximes. This factor enables a freedom of choiceover the chemical group for the desired reversible conjugation reaction.Contrary, the alcohols are good candidates for reversible conjugationreaction via the formation of acetals and ketals as well, but lack inchemoselectivity since they are present in a large quantity on theglycosidic structure.

For the formation of an irreversible and stable bond the carboxylic acidis the most suitable since it can form amides and esters with the commontools used in peptide chemistry (e.g. reaction with amines viacarbodiimide mediated amide formation).

TABLE 1 Functional groups that are available for saponin conjugationreactions Functional Group Pros Cons Alcohol Suitable for reversibleacetal/ketal Acetal/ketal formation without (Diols) formationchemoselectivity Suitable for ester formations with Ester formationwithout activated carboxylic acids chemoselectivity Aldehyde Suitablefor chemoselective Not suitable for acetal formation in reversiblehydrazone formation with the presence of unprotected hydrazides saponinsugar diols Suitable for chemoselective reversible imine formation withamines Suitable for chemoselective reversible oxime formation withhydroxylamines Alkene Suitable for chemoselective Not suitable forreversible irreversible radical reactions conjugation reactions Notsuitable for reactions involving a hydrogenation step CarboxylicSuitable for chemoselective amide/ Not suitable for reversible acidester formation with amines and conjugation reactions under mildalcohols after activation conditions

Regarding an ideal EMCH spacer length for conjugation to a polymericstructure, computer simulation (PerkinElmer, ChemBio3D, Ver.13.0.0.3015) shows that the maleimide group on SO1861-EMCH is located atthe periphery of the molecule and thus should be accessible for thiolbearing polymeric structures (FIG. 27-1).

As a polymeric structure, a G4-dendron (PFd-G4-Azide-NH-BOC, PolymerFactory) with 16 functional amino end groups and an azido group at thefocal point was utilized for the conjugation to SO1861 (FIG. 24-1). Theadvantage of using a dendron over a dendrimer is the focal point thatthe dendron structure is exhibiting.

Another approach for the development of a SO1861 scaffold among thediscussed polymer, and protein approach is the poly(SO1861) approach.The idea of this approach is to generate a polymer that consists ofSO1861 molecules only, with pH sensitive cleavable bonds that releasethe SO1861. In addition, the poly(SO1861) should be able to performconjugation reactions to toxins and biopolymers. The main goal with thisapproach is to keep it as simple and cost effective as possible. Since aprotocol for the generation of acid cleavable SO1861 has been developedalready (SO1861-EMCH approach) it would be interesting to see if it ispossible to polymerize the SO1861-EMCH through simple addition of apolymerization initiator without further modifying the SO1861 oridentifying other conjugation sites on the SO1861 molecule. In the past,several papers have discussed the polymerization of maleimide groups byusing radical initiators which attack the double bond of the maleimidegroup and thus initiate a radical polymerization along the double bondsof the maleimides. Since SO1861-EMCH reveals a maleimide group in itsstructure this group could potentially be explored for radicalpolymerization reactions to yield a poly(SO1861) with acid cleavablefunction. If the polymerization reaction has a reasonable reaction timethe generated SO1861 polymers could be quenched with a radical quencherthat not only quenches the reaction but also generates a functionalgroup for toxin or biopolymer conjugation. Here, the system of ammoniumpersulfate (APS) and tetramethylethylenediamine (TMEDA) is indicated inan exemplary way as radical generator and aminopropanethiol serves as amodel radical quencher. Using aminopropanethiol as a quencher exemplary,the generated amine group could be specifically further modified to aclick-able group or being used to directly conjugate the poly(SO1861) toa toxin.

Another approach for the development of a SO1861 scaffold is the DNAapproach. The idea of this approach is to utilize the concept of theso-called DNA-origami (Kolb et al, 2004; Bird et al, 1988). DNA-origamias the polymeric or assembled polymeric structure to conjugate saponinsto it, can offer several inherent advantages including stability,scalability, and precise control of the final size and shape of theresulting DNA-saponin scaffold. Since these DNA nanocarriers arecomprised of natural DNA, they are biocompatible and do not showtoxicity to living cells, and can ease the release of cargo frominternal cellular compartments. The multivalency of such a structure canfurther allow fine-tuning targeting capabilities and high capacity for avariety of payloads such as fluorophores and toxins. Thus, in thisapproach DNA strands are identified that offer chemical functionalgroups on the 3′ and 5′ endings respectively, and that are able tohybridize only in certain wanted areas of the sequence that allow acontrol over the final shape of the construct. The chemical groupsshould be utilized to couple saponins, for instance though a thiol-enereaction between the already developed SO1861-EMCH and a thiol group onone of the 3′ and 5′ DNA strands. The complementary DNA strand can offera click function group that can be used for coupling to a targetedtoxin. The concept is illustrated in FIG. 23-1.

A similar approach is imaginable by using a specific peptide sequenceinstead of DNA strands that is able to bind and release saponins andthat can be polymerized forming a large poly(peptide)-like structure. Inthis approach, a peptide sequence has been identified and purchased thathas a length fitting the calculated size of a SO1861-EMCH molecule, thatoffers a cysteine residue in the middle of the sequence, and thatobtains an amine group at both the N-terminus and C-terminus. Thecysteine residue can be utilized to conjugate SO1861-EMCH via athiol-ene reaction of the maleimide group of SO1861-EMCH and the thiolgroup of the cysteine residue. The two amine groups can be utilized topolymerize the peptide-SO1861 conjugate with a suitable crosslinker.

Example 8 SO1861-BNA Oligo Conjugation

HSP27 BNA oligo disulfide (1.10 mg, 0.187 μmol) was dissolved in 20 mMNH₄HCO₃ with 1.0 mM TCEP (500 μL) and the mixture was shaken for 1 minand left standing at room temperature. After 1 hour the reaction mixturewas filtered by using a centrifugal filter with a molecular weightcut-off of 3000 Da (14000×g for 30 min). The residue solution wasdiluted with 20 mM NH₄HCO₃ with 1.0 mM TCEP (500 μL) and the resultingmixture was filtered again under the same conditions described above.The residue solution was diluted with 20 mM NH₄HCO₃/acetonitrile (3:1,v/v, 1.00 mL) and the resulting mixture was added to SO1861-EMCH (3.54mg, 0.375 μmol). The reaction mixture was shaken for 1 min and leftstanding at room temperature. After 10 min the reaction mixture wassubjected to preparative LC-MS.^(4A) Fractions corresponding to theproduct were immediately pooled together, frozen and lyophilizedovernight to give the title compound (1.25 mg, 85%) as a white fluffysolid. Purity based on LC-MS 100%.

LRMS (m/z): 1561 [M-5]⁵⁻, 1951 [M-4]⁴⁻

LC-MS r.t. (min): 2.46^(6B)

Dendron(SO1861)⁴-BNA Oligo Conjugation

HSP27 BNA oligo disulfide (1.1 mg, 0.187 μmol) was dissolved in 20 mMNH₄HCO₃ with 1.0 mM TCEP (500 μL) and the mixture was shaken for 1 minand left standing at room temperature. After 1 hour the reaction mixturewas filtered by using a centrifugal filter with a molecular weightcut-off of 3000 Da (14000×g for 30 min). The residue solution wasdiluted with 20 mM NH₄HCO₃ with 1.0 mM TCEP (500 μL) and the resultingmixture was filtered again under the same conditions described above.The residue solution was diluted with 20 mM NH₄HCO₃/acetonitrile (3:1,v/v, 1.0 mL) and the resulting mixture was added todendron(SO1861)4-maleimide1 (3.54 mg, 0.375 μmol). The reaction mixturewas shaken for 1 min and left standing at room temperature. After 10 minthe reaction mixture was subjected to preparative LC-MS.^(4A) Fractionscorresponding to the product were immediately pooled together, frozenand lyophilized overnight to give the title compound (1.25 mg, 85%) as awhite fluffy solid. Purity based on LC-MS 94%

LRMS (m/z): 1896 [M-8]⁸⁻, 2167 [M-7]⁷⁻

LC-MS r.t. (min): 3.77^(6B)

Cell Culture

Cells were seeded in DMEM (PAN-Biotech GmbH) supplemented with 10% fetalbovine serum (PAN-Biotech GmbH) and 1% penicillin/streptomycin(PAN-Biotech GmbH), in a 96 well plate at 5,000 c/w in 100 μL/well andincubated overnight at 37° C. and 5% CO₂. The next day samples wereprepared in DMEM and cells were treated.

Gene Silencing

RNA isolation and Qper analysis was performed according to standardprocedures and protocols.

HSP27 primers: F: R:

HSP27BNA Oligo

HSP27BNA(-thiol) oligos (sequence 5′-GGCacagccagtgGCG-3′) (Zhang et al.,2011) were ordered at Bio-synthesis Inc. (Lewisville, Tex.)

Results

BNAoligo, antisense BNA oligo targeting the mRNA transcript of thecancer target (upregulated in cancer cells), heat shock protein 27(HSP27BNA) was conjugated to SO1861-EMCH (HSP27BNA-L-SO1861) ordendron(-L-SO1861)⁴ (HSP27BNA-dendron(-L-SO1861)⁴) and co-administratedto an A431 cancer cell line, according to the invention. As readout,gene silencing of HSP27 mRNA in A431 cells was determined. This revealedthat HSP27BNA-L-SO1861 treatment resulted in an improvement of HSP27gene silencing activity compared to the HSP27BNA alone, whereas theactivity of HSP27BNA-dendron(-L-SO1861)⁴ (4 SO1861 molecules/BNA) iseven stronger (3-fold) compared to the gene silencing activity ofHSP27BNA alone (FIG. 1-3). This shows that conjugation of 1 or moreSO1861 molecules improves the gene silencing activity of the therapeuticBNA oligo nucleotide due to the enhancement of SO1861-mediated endosomalescape and cytoplasmic delivery of the antisense BNA.

Example 9

SO1861 was conjugated (labile) via cysteine residues (Cys) and dianthin(protein toxin) was conjugated (stable) via lysine residues (Lys) tocetuximab (monoclonal antibody recognizing and binding human EGFR),resulting in the production of:Cetuximab-(Cys-L-SO1861)^(3,9)(Lys-S-dianthin)². The conjugate wastested in a A431 (EGFR⁺⁺) xenograph mouse tumor model for EGFR tumortargeted cell killing as illustrated in FIG. 9-4. Dosings started at day12 when tumors reached ˜150 mm³ in size and tumor volume was determinedafter every dosing. Mice (n=3) were treated (intraperitoneal; i.p.; doseescalation) at day 12: 0.5 mg/kg; dayl5: 1 mg/kg and day24: 1.5 mg/kgwith cetuximab-(Cys-L-SO1861)^(3,9)(Lys-S-dianthin)² orcetuximab-(Lys-S-dianthin)¹′⁶. At day 26, compared to the control group,tumor volume reduction could be observed in the tumor bearing micetreated with cetuximab-(Cys-L-SO1861)^(3,9)(Lys-S-dianthin)² (FIG.1-4A). This shows that labile conjugation of SO1861 to anantibody-protein toxin (stable) conjugate can enhance the targetedtherapeutic efficacy of the tumor targeted antibody-protein toxin,thereby inducing a more effective tumor targeted therapy.

Next, SO1861 was conjugated (labile) via cysteine residues (Cys) anddianthin (protein toxin) was conjugated (labile) via lysine residues(Lys) to cetuximab (monoclonal antibody recognizing and binding humanEGFR), resulting in the production of:Cetuximab-(Cys-L-SO1861)^(3,9)(Lys-L-dianthin)². The conjugate wastested in a A431 (EGFR⁺⁺) xenograph mouse tumor model for EGFR tumortargeted cell killing as illustrated in FIG. 9-4. Dosings started at day12 when tumors reached ˜150 mm³ in size and tumor volume was determinedafter every dosing. Mice (n=3) were treated (intraperitoneal; i.p.; doseescalation) at day 12: 0.5 mg/kg; dayl5: 1 mg/kg, day24: 1.5 mg/kg withcetuximab-(Cys-L-SO1861)^(3,9)(Lys-L-dianthin)² orcetuximab-(Lys-L-dianthin)¹⁶. This revealed that after 35 days comparedto the control, tumor bearing mice treated withcetuximab-(Cys-L-SO1861)^(3,9)(Lys-L-dianthin)² showed tumor growthinhibition (FIG. 1-4B). When mice (n=3; were treated (intravenous, i.v.;dose escalation) day 12: 0.5 mg/kg; dayl5: 1 mg/kg, dayl8: 2 mg/kg,day24: 2.5 mg/kg with thecetuximab-(Cys-L-SO1861)^(3,9)(Lys-L-dianthin)² according to theinvention also tumor growth inhibition could be observed compared to thecontrol (data represents 1 mice, since 2 mice died during thetreatments). This shows that labile conjugation of SO1861 to anantibody-protein toxin (labile) conjugate can enhance the targetedtherapeutic efficacy of the tumor targeted antibody-protein toxin,thereby inducing a more effective tumor targeted therapy.

Next, SO1861-EMCH was conjugated via cysteine residues (Cys) tocetuximab (monoclonal antibody recognizing and binding human EGFR), witha DAR 3,9 and the antisense HSP27BNA oligo nucleotide (targeting andinducing degradation of the onco-target hsp27 mRNA (gene silencing) incancer cells) via a labile (L) linker to the lysine residues (Lys) ofthe antibody, with a DAR 1,8 resulting in the production ofcetuximab-(Cys-L-SO1861)^(3,9)(Lys-L-HSP27BNA)^(1,8).Cetuximab-(Cys-L-SO1861)^(3,9)(Lys-L-HSP27BNA)^(1,8)was tested in a A431xenograph ‘nude’ mouse tumor model for EGFR-mediated tumor targetedHSP27 gene silencing, according to the invention as illustrated in FIG.10-4. Dosing started at day 12 when tumors reached ˜150 mm³ in size andHSP27 mRNA expression was determined. For this, tumor samples werecollected at 72h after the first dosing and analysed for HSP27 geneexpression levels compared to cellular control mRNA expression levels(reference genes). Tumor bearing mice (n=3) treated (intraperitoneal;i.p.) with 30 mg/kg cetuximab-(Cys-L-SO1861)^(3,9)(Lys-L-HSP27BNA)^(1,8)showed after 1 dosing 40% reduction in HSP27 mRNA expression in thetumors compared to single dosing of cetuximab-(Cys-L-SO1861)^(3,8) orcetuximab-(Lys-L-HSP27BNA)¹′⁵ (FIG. 2-4). Compared to the tumor of thevehicle control a reduction of 25% HSP27 gene expression was observed.This shows and enables that conjugation of SO1861 and HSP27BNA to thesame targeting antibody, according to the invention, efficiently inducesSO1861-mediated enhanced cytoplasmic delivery of a therapeutic antisenseoligo nucleotide in solid tumors of tumor bearing mice, inducing tumortargeted gene silencing. In another example, a trifunctional linkerscaffold was designed and produced with 3 specific chemical end groupsfor conjugation with SO1861 on one arm and the HSP27BNA on the other armto produce SO1861-L-trifunctional linker-L-HSP27BNA. Next,SO1861-L-trifunctional linker-L-HSP27BNA was conjugated with its thirdarm to cysteine residues (Cys) of the anti-EGFR antibody, cetuximab(cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA)³′⁷) and testedin a A431 xenograph ‘nude’ mouse tumor model for EGFR-mediated tumortargeted gene silencing activity, according to the invention asillustrated in FIG. 11-4. Dosings started at day 12 when tumors reached˜150 mm³ in size and HSP27 mRNA expression was determined. For this,tumor samples were collected at 72h after the first dosing and analysedfor HSP27 gene expression levels compared to cellular control mRNAexpression levels (reference genes). This revealed that 1 dosing of 30mg/kg cetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA)³′⁷resulted in a 40% reduction in HSP27 gene expression in the tumorscompared to single dosing of 25 mg/kg cetuximab-(Cys-L-SO1861)^(3,8) or25 mg/kg cetuximab-(Lys-L-HSP27BNA)⁴ mono therapies (FIG. 3-4). Comparedto the vehicle control tumors, a reduction of 25% HSP27 gene expressionwas observed in tumor bearing mice treated with 1 dosing ofcetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA)³⁷. This showsand enables that cetuximab-Cys-(SO1861-L-trifunctionallinker-L-HSP27BNA)³′⁷ efficiently induces SO1861-mediated enhancedcytoplasmic delivery of a therapeutic antisense oligo nucleotide in asolid tumor of tumor bearing mice, inducing targeted gene silencing, invivo.

Example 10

In another example according to the invention, SO1861 (labile) and theprotein toxin, dianthin (labile or stable) were conjugated to the HER2targeting antibody, trastuzumab.Trastuzumab-(Cys-L-SO1861)^(3,8)(Lys-L-dianthin)^(1,7) ortrastuzumab-(Cys-L-SO1861)^(3,8)(Lys-S-dianthin)^(1,7), were producedand tested for enhanced cell killing in SK-BR-3 (HER2⁺⁺) and MDA-MB-468(HER2⁻) cells as illustrated in FIG. 9-4. Both,trastuzumab-(Cys-L-SO1861)^(3,8)(Lys-L-dianthin)^(1,7) (IC50=0.8 nM) andtrastuzumab-(Cys-L-SO1861)^(3,8)(Lys-S-dianthin)^(1,7) (IC50=0.8 nM)efficiently induces cell killing of SK-BR-3 cells (HER2⁺⁺) (FIG. 4-4A).This was not observed in SK-BR-3 cells treated with trastuzumab,trastuzumab-(Lys-L-dianthin)¹⁷, trastuzumab-(Lys-S-dianthin)¹⁷ ortrastuzumab-(L-SO1861)^(3,8) alone (FIG. 4-4A). In MDA-MB-468 cells(HER2⁻) no cell killing activity can be observed for any of theconjugates, according to the invention (FIG. 4-4B). This shows thatconjugation of SO1861 to an HER targeting antibody-protein toxinconjugate, efficiently induces SO1861-mediated enhanced cytoplasmicdelivery of the protein toxin in the target cell resulting in targetcell death.

In another example according to the invention, SO1861 (labile) and theprotein toxin, dianthin (labile or stable) were conjugated to the EGFRtargeting antibody, cetuximab.Cetuximab-(Cys-L-SO1861)^(3,9)(Lys-L-dianthin)² orcetuximab-(Cys-L-SO1861)^(3,9)(Lys-S-dianthin)², was tested for enhancedcell killing in A431 cells (EGFR⁺⁺) and A2058 cells (EGFR⁻) asillustrated in FIG. 9-4. Both,cetuximab-(Cys-L-SO1861)^(3,9)(Lys-L-dianthin)² (IC50=0.3 nM) andcetuximab-(Cys-L-SO1861)^(3,8)(Lys-S-dianthin)¹⁷ (IC50=0.3 nM) showedenhanced cell killing in A431 cells (EGFR⁺⁺) compared tocetuximab-(Lys-L-dianthin)¹′⁶ (IC50=2 pM), cetuximab-(Lys-S-dianthin)¹′⁶(IC5=2 pM) alone (FIG. 4-4C). In A2058 cells (EGFR⁻) the combinationaccording to the invention did not show any cell killing activity(IC50>200 nM; FIG. 4-4D). This shows that conjugation of SO1861 to anEGFR targeting antibody-protein toxin conjugate, efficiently enhancesSO1861-mediated cytoplasmic delivery of the protein toxin in the targetcell resulting in enhanced target cell death.

Example 11

In another example according to the invention, SO1861 (labile) and theHSP27BNA oligo (labile) were conjugated to the EGFR targeting antibody,cetuximab. Cetuximab-(Cys-L-SO1861)^(3,8)(Lys-L-HSP27BNA)^(3,8) wastested for enhanced HSP27 gene silencing in A431 cells (EGFR⁺⁺) andA2058 (EGFR⁻) cells, according to the invention as illustrated in FIG.10-4. Cetuximab-(Cys-L-SO1861)^(3,8)(Lys-L-HSP27BNA)^(3,8) efficientlyinduces HSP27 gene silencing in A431 cells (IC50=3 nM) compared tocetuximab, cetuximab-(Lys-L-HSP27BNA)³′⁹ orcetuximab-(Cys-L-SO1861)^(3,8) alone

(FIG. 5-4A). In A2058 cells (EGFR⁻) no gene silencing activity can beobserved with cetuximab-(Cys-L-SO1861)^(3,8)(Lys-L-HSP27BNA)^(3,8)(IC50>100 nM; FIG. 5-4B). This shows and enables that conjugation ofSO1861 and HSP27BNA to the same targeting antibody, according to theinvention, efficiently induces SO1861-mediated enhanced cytoplasmicdelivery of a therapeutic antisense oligo nucleotide in the targetcells, inducing targeted gene silencing.

In another example according to the invention, SO1861 (labile) and theHSP27BNA oligo (labile) were conjugated to the HER2 targeting antibody,trastuzumab. Trastuzumab-(Cys-L-SO1861)^(3,8)(Lys-L-HSP27BNA)³⁵ wastested for enhanced HSP27 gene silencing in SK-BR-3 cells (HER2⁺⁺)cells, according to the invention as illustrated in FIG. 10-4.Trastuzumab-(Cys-L-SO1861)^(3,8)(Lys-L-HSP27BNA)^(3,5) efficientlyinduces HSP27 gene silencing in SK-BR-3 cells (IC50=9 nM) compared totrastuzumab-(Lys-L-HSP27BNA)^(4,4) alone (FIG. 6-4). This shows andenables that conjugation of SO1861 and HSP27BNA to an HER2 targetingantibody, according to the invention, efficiently inducesSO1861-mediated enhanced cytoplasmic delivery of a therapeutic antisenseoligo nucleotide in the target cells, inducing targeted gene silencing.

In another example, cetuximab-Cys-(SO1861-L-trifunctionallinker-L-HSP27BNA)³′⁷ was tested for enhanced HSP27 gene silencing inA431 (EGFR⁺⁺) and A2058 (EGFR⁻) cells according to the invention asillustrated in FIG. 11-4. Cetuximab-Cys-(SO1861-L-trifunctionallinker-L-HSP27BNA)³′⁷ efficiently induces HSP27 gene silencing in A431cells (IC50=2 nM) compared to Cetuximab-(Lys-L-HSP27BNA)⁴ orCetuximab-(Cys-L-SO1861)^(3,7) alone (FIG. 7-4A). In A2058 cells (EGFR⁻)gene silencing activity was only observed at high (>80 nM)concentrations of Cetuximab-Cys-(SO1861-L-trifunctionallinker-L-HSP27BNA)^(3,7) (IC50=100 nM; FIG. 7-4B). This shows andenables that in high EGFR expressing cellscetuximab-Cys-(SO1861-L-trifunctional linker-L-HSP27BNA)³′⁷ efficientlyinduces SO1861-mediated enhanced cytoplasmic delivery of a therapeuticantisense oligo nucleotide in the target cells, inducing targeted genesilencing.

Example 12

FIG. 8-4A-D displays the relative cell viability when trastuzumab (FIG.8-4A), cetuximab (FIG. 8-4B) or T-DM1 (FIG. 8-4C), unconjugated proteintoxins, saporin, dianthin and saporin conjugated to a (non-cell binding)IgG antibody (FIG. 8-4D) are administered to various cancer cell linesSK-BR-3, JIMT-1, MDA-MB-468, A431, CaSki, HeLa, A2058.

Trastuzumab and cetuximab do not or hardly influence cell viability whenexposed to most of the cell lines, with some effect on cell growthinhibition via blocking the function of the HER2 growth factor receptorwhen trastuzumab is exposed to SK-BR-3 cells at relatively high dose andwith some effect on cell growth inhibition via blocking the function ofthe EGFR growth factor receptor when cetuximab is exposed to MDA-MB-468cells at relatively high dose.

TDM-1, or ado-trastuzumab emtansine, is a targeted therapy approved bythe U.S. Food and Drug Administration to treat: HER2-positive metastaticbreast cancer that has previously been treated with Herceptin (chemicalname: trastuzumab) and taxane chemotherapy; early-stage HER2-positivebreast cancer after surgery if residual disease was found afterneoadjuvant (before surgery) treatment with Herceptin and taxanechemotherapy. The TDM-1 is a combination of Herceptin (Trastuzumab) andthe chemotherapy medicine emtansine. FIG. 8-4C shows that the TDM-1results in decreased cell viability for all cell lines tested at >1000pM concentrations

The free toxins saporin and dianthin and the toxin saporin coupled to acontrol IgG with no affinity for any of the cell surface molecules onthe cell lines tested, do not or hardly have any influence on cellviability over a wide range of concentrations toxin tested, up to100.000 pM (FIG. 8-4D).

Example 13 (Example 1 Invention 5)

The 1 target 2-components system (1T2C) is the combination treatment ofmAb1-protein toxin and mAb1-SO1861, as illustrated in FIG. 13-5.SO1861-EMCH was conjugated via cysteine residues (Cys) and HSP27BNAoligo was conjugated via lysine residues to cetuximab (monoclonalantibody recognizing and binding human EGFR), both with a DAR 4resulting in the production of 2 conjugates: cetuximab-(Cys-L-SO1861)⁴and cetuximab-(Lys-L-HSP27BNA)⁴. The combination ofcetuximab-(Cys-L-SO1861)⁴ (intraperitoneal administration, (i.p.)) andcetuximab-(Lys-L-HSP27BNA)⁴ (intravenous administration, (i.v.)) wastested in a A431 xenograph ‘mouse tumor model for EGFR tumor targetedgene silencing activity. Dosings started at day 12 when tumors reached˜150 mm³ in size and tumor samples were collected at 72h after the firstdosing and analysed for HSP27 gene expression compared to control genemRNA expression levels (reference genes). This revealed that 1 dosing of50 mg/kg cetuximab-(Cys-L-SO1861)⁴+25 mg/kg cetuximab-(Lys-L-HSP27BNA)⁴resulted in a 50% reduction in HSP27 gene expression in the A431 tumorscompared to single dosing of cetuximab-(Cys-L-SO1861)⁴ orcetuximab-(Lys-L-HSP27BNA)⁴ mono therapies (FIG. 1-5). Compared to thevehicle control tumors, a reduction of 40% HSP27 gene silencing wasobserved. This shows and enables that the combination ofcetuximab-conjugated SO1861+cetuximab-conjugated HSP27BNA oligo,according to the 1T2C invention, induces efficient targeted delivery ofa therapeutic antisense oligo nucleotide in the cytoplasm of solid tumorcells, thereby inducing tumor targeted gene silencing, in vivo.

Next, SO1861-EMCH was conjugated via cysteine residues (Cys) totrastuzumab (monoclonal antibody recognizing and binding human HER2),with a DAR 4 resulting in the production of trastuzumab-(Cys-L-SO1861)⁴.The combination of trastuzumab-(Cys-L-SO1861)⁴ and trastuzumab-saporin(trastuzumab protein toxin conjugate) was tested in a mouse tumor model(patient derived xenograph tumor model, PDX) with high HER2 expressionlevels and resistant for trastuzumab mono therapy. The combination,according to the 1T2C invention of 40 mg/kg trastuzumab-(Cys-L-SO1861)⁴(intraperitoneal administration, (i.p.))+0.03 (Dayl, 8)/0.02 (Day 15,22, 30, 36,43) mg/kg trastuzumab-saporin (intravenous administration,(i.v.)) revealed strong tumor growth inhibition compared to the vehiclecontrol and the 40 mg/kg trastuzumab-(Cys-L-SO1861)⁴ or 0.03/0.02 mg/kgtrastuzumab-saporin mono therapies (FIG. 2-5). Besides, in tumor bearingmice that were treated with a lower dosing combination (40 mg/kgtrastuzumab-(Cys-L-SO1861)⁴+0.01 mg/kg trastuzumab-saporin) no tumorgrowth inhibiting activity was observed (FIG. 2-5). This shows andenables that the 1T2C combination of trastuzumab conjugatedSO1861+trastuzumab conjugated protein toxin induces efficient targeteddelivery of a therapeutic protein toxin in the cytoplasm of solid tumorcells, thereby inducing tumor cell death and tumor growth inhibition, invivo.

Example 14

The 1 Target 2-Components System (1T2C) is the Combination Treatment ofmAb1-SO1861 and mAb1-Protein Toxin (FIG. 13-5)

SO1861-EMCH was conjugated via cysteine residues (Cys) to cetuximab(monoclonal antibody recognizing and binding human EGFR), with a DAR 3,7(cetuximab-(Cys-L-SO1861)^(3,7)). Cetuximab-(Cys-L-SO1861)^(3,7) wastitrated on a fixed concentration of 10 pM cetuximab-saporin (cetuximab,conjugated to the protein toxin, saporin) and targeted protein toxinmediated cell killing on EGFR expressing cells (A431, EGFR++; CaSKi,EGFR⁺) was determined. This revealed strong cell killing at lowconcentrations of cetuximab-(Cys-L-SO1861)^(3,7) (A431: IC50=0.6 nM andCaski IC50=1 nM; FIG. 5A, 3-5B) whereas cetuximab,cetuximab-(Cys-L-SO1861)^(3,7) or cetuximab+10 pM cetuximab-saporincould not induce any cell killing activity in EGFR expressing cells.This shows that cetuximab conjugated SO1861 efficiently enhancesendosomal escape of the cetuximab conjugated protein toxin (atnon-effective concentrations), thereby inducing cell killing of EGFRexpressing cells. The cell killing activity in A431 is more effectivecompared to CaSki correlating with EGFR expression levels in these celllines. EGFR receptor binding competition between both conjugates withinthe 1T2C is also observed when cetuximab-(Cys-L-SO1861)^(3,7)concentrations increase, cell killing activity declines due tooutcompeting receptor binding and internalization of cetuximab-saporin(FIG. 3-5A, 3-5B).

Next, cetuximab-saporin was titrated on a fixed concentration of 75 nMcetuximab-(Cys-L-SO1861)^(3,7) and targeted protein toxin mediated cellkilling on EGFR expressing cells was determined. This revealed that 75nM cetuximab-(Cys-L-SO1861)^(3,7) in combination with low concentrationscetuximab-saporin induced already efficient cell killing in EGFRexpressing cells (A431: IC50=0.4 pM; and CaSKi: (IC50=2 pM; FIGS. 3-5Cand 3-5D), whereas cetuximab-saporin alone or cetuximab-saporin+75 nMcetuximab showed cell killing only at high concentrationscetuximab-saporin (IC50=40 pM, IC50=1000 pM, resp.) in both cell lines(FIG. 3-5C, 3-5D). All this shows that relatively low concentrations ofcetuximab-saporin can be effective and induce cell killing only incombination with low cetuximab-SO1861 concentrations in high EGFRexpressing cells. The receptor competition between both conjugateswithin the 1T2C system is also observed in the cetuximab-toxin titrationtreatments when the cell killing activity of cetuximab-saporin with andwithout 75 nM cetuximab was compared (FIG. 3-5C, 3-5D).

Next, cetuximab-(Cys-L-SO1861)^(3,7) was titrated on a fixedconcentration of 10 pM cetuximab-saporin and targeted protein toxinmediated cell killing on low EGFR expressing cells or cells without EGFRexpression (HeLa, EGFR^(+/−); A2058, EGFR⁻) was determined. Cells withlow (HeLa) or no (A2058) EGFR expression were not sensitive at all forany combination of cetuximab-(Cys-L-SO1861)^(3,7)+10 pMcetuximab-saporin (HeLa: IC50>1000 nM; A2058: IC50>1000 nM; FIG. 4-5A,4-5B). This shows that in the absence of sufficient EGFR receptorexpression, effective intracellular delivered SO1861 concentrations arenot optimal (threshold) to induce endosomal protein toxin escape andtoxin-mediated cell killing. Next, cetuximab-saporin was titrated on afixed concentration of 75 nM cetuximab-(Cys-L-SO1861)^(3,7) and targetedprotein toxin mediated cell killing on low (HeLa) or no (A2058) EGFRexpressing cells was determined. Low EGFR expressing cells (HeLa) showedcell killing only at high cetuximab-saporin concentrations incombination with 75 nM cetuximab-(Cys-L-SO1861)^(3,7) (HeLa: IC50=60pM), FIG. 4-5C), whereas A2058 cells (EGFR⁻) are not sensitive at any ofthe tested concentrations (A2058: IC50>10.000 pM; FIG. 4-5D). All thisshows that cells with low or no EGFR receptor expression are notsusceptible for the combination ofcetuximab-(Cys-L-SO1861)^(3,7)+cetuximab-saporin, due to a lack ofsufficient EGFR receptor that facilitates the antibody-mediated deliveryof sufficient SO1861 within the endolysosomal compartments, tofacilitate the escape of the protein toxin.

Next, SO1861-EMCH was conjugated via cysteine residues (Cys) totrastuzumab (monoclonal antibody recognizing and binding human HER2,with a DAR 4, (trastuzumab-(Cys-L-SO1861)⁴). Trastuzumab-(Cys-L-SO1861)⁴was titrated on a fixed concentration of 50 pM trastuzumab-saporin(trastuzumab, conjugated to the protein toxin, saporin) and targetedprotein toxin mediated cell killing on HER2 expressing cells (SK-BR-3,HER2⁺⁺) was determined. This revealed strong cell killing at lowconcentrations of trastuzumab-(Cys-L-SO1861)⁴ (SK-BR-3: IC50=0.8 nM;FIG. 5-5A) whereas equivalent concentrations trastuzumab,trastuzumab-(Cys-L-SO1861)⁴ or trastuzumab+50 pM trastuzumab-saporincould not induce any cell killing activity in HER2 expressing cells.This shows that trastuzumab conjugated SO1861 efficiently enhancesendosomal escape of the trastuzumab conjugated protein toxin (atnon-effective concentrations), thereby inducing cell killing of HER2expressing cells. The receptor competition between both conjugateswithin the 1T2C is also observed when trastuzumab-(Cys-L-SO1861)⁴concentrations increase, cell killing activity declines due tooutcompeting receptor binding and internalization of trastuzumab-saporin(FIG. 5-5A).

Next, trastuzumab-saporin was titrated on a fixed concentration of 2.5nM trastuzumab-(Cys-L-SO1861)⁴, according to the invention and targetedprotein toxin mediated cell killing on HER2 expressing cells wasdetermined. This revealed that 2.5 nM trastuzumab-(Cys-L-SO1861)⁴ incombination with low concentrations trastuzumab-saporin induced alreadyefficient cell killing in HER2 expressing cells (SK-BR-3: IC50=2 pM;FIG. 5-5B), whereas trastuzumab-saporin alone or trastuzumab-saporin+2.5nM trastuzumab showed cell killing only at high concentrationstrastuzumab-saporin (FIG. 5-5B). All this shows that relatively lowconcentrations of trastuzumab-saporin can be effective and induce cellkilling only in combination with low trastuzumab-(Cys-L-SO1861)⁴concentrations in high HER2 expressing cells.

Next, trastuzumab-(Cys-L-SO1861)⁴ was titrated on a fixed concentrationof 50 pM trastuzumab-saporin, according to the invention and targetedprotein toxin mediated cell killing on low HER2 expressing cells (A431HER2^(+/−)) or cells without HER2 expression (JIMT-1: HER2⁺; MDA-MB-468:HER2⁻) was determined. Cells with low or no HER2 expression were notsensitive at all for any combination of trastuzumab-(Cys-L-SO1861)⁴+50pM trastuzumab-saporin (JIMT-1: IC50>1000 nM; MDA-MB-468: IC50>1000 nM;FIG. 6-5A, 6-5B). This shows that in the absence of sufficient HER2receptor expression, effective intracellular delivered SO1861concentrations are not optimal (threshold) to induce endosomal proteintoxin escape and toxin-mediated cell killing. Next, trastuzumab-saporinwas titrated on a fixed concentration of 2.5 nMtrastuzumab-(Cys-L-SO1861)⁴ and targeted protein toxin mediated cellkilling on low or no HER2 expressing cells was determined. Low HER2expressing cells (JIMT-1) showed cell killing only at hightrastuzumab-saporin concentrations in combination with 2.5 nMtrastuzumab-(Cys-L-SO1861)⁴ (JIMT-1: IC50>10.000 pM; FIG. 6-5C), whereasMDA-MB-468 cells (HER2⁻) are not sensitive at any of the testedconcentrations (MDA-MB-468: IC50>10.000 pM; FIG. 6-5D).

All this shows that cells with low or no HER2 receptor expression arenot susceptible for the combination oftrastuzumab-(Cys-L-SO1861)^(3,7)+trastuzumab-saporin, due to a lack ofsufficient HER2 receptor that facilitates the antibody-mediated deliveryof sufficient SO1861 within the endolysosomal compartments, tofacilitate the escape of the protein toxin.

Example 15

In order to show that the activity of the 1T2C system is driven by theacidification of the endolysosomal compartments, the 1T2C system,according to the invention was tested in combination with an endosomalacidification inhibitor, chloroquine. Trastuzumab-saporin was titratedin combination with 5 nM trastuzumab-(Cys-L-SO1861)⁴ in combination withor without chloroquine. Trastuzumab-saporin+5 nMtrastuzumab-(Cys-L-SO1861)⁴ showed a strong cell killing activity inhigh HER2 expressing cells (SK-BR-3, HER2⁺⁺; IC50=0.2 pM;), however,trastuzumab-saporin+5 nM trastuzumab-(Cys-L-SO1861)⁴+0.5 pM chloroquineresulted in strong inhibition of the 1T2C cell killing activity inSK-BR-3 (HER2⁺⁺) cells (IC50=40 pM). This shows that activity of theantibody conjugated SO1861 is reduced/blocked when acidification ofendoslysomes is prohibited (FIG. 7-5A). Same results were derived withthe 1T2C combination, according to the invention of cetuximab-saporin+5nM cetuximab-(Cys-L-SO1861)^(3,8) (IC50=1 pM) compared withcetuximab-saporin+5 nM cetuximab-(Cys-L-SO1861)^(3,8)+0.5 pM chloroquine(IC50=200 pM) in EGFR expressing cells (A431, EGFR++; FIG. 7-5B).

Example 16

The 1 target 2-components system (1T2C) can also be the combinationtreatment of mAb1-SO1861 and mAb1-antisense BNA oligo nucleotide asillustrated in FIG. 14-5. For this we used an antisense BNAoligonucleotide against the mRNA of a cancer specific target gene(upregulated in cancer cells), heat shock protein 27 (HSP27). Uponrelease into the cytoplasm the antisense BNA recognizes and binds themRNA encoding for HSP27, targeting the mRNA for destruction therebydepleting the HSP27 mRNA expression within the cancer cell. HSP27BNA wasconjugated to cetuximab with a DAR4 (Cetuximab-(Lys-L-HSP27BNA)⁴) andtested in combination with cetuximab-(Cys-L-SO1861)^(3,8) for enhancedHSP27 gene silencing activity in EGFR expressing cells (A431, EGFR⁺⁺)and non-expressing cells (A2058, EGFR), according to the invention (FIG.14-5). Cetuximab-(Cys-L-SO1861)^(3,8)+100 nM cetuximab-(Lys-L-HSP27BNA)⁴showed strong HSP27 gene silencing in EGFR expressing cells (A431:IC50=nM, FIG. 8-5A), whereas cetuximab-(Cys-L-SO1861)^(3,8) alone didnot show any gene silencing activity. In A2058 cells (EGFR⁻) no genesilencing activity was observed in the 1T2C combination (FIG. 8-5B).Next, cetuximab-(Lys-L-HSP27BNA)⁴+76.9 nM Cetuximab-(Cys-L-SO1861)^(3,8)show strong HSP27 gene silencing activity in EGFR expressing cells(A431: IC50=4 nM, FIG. 8-5C), whereas cetuximab-(Lys-L-HSP27BNA)⁴ orcetuximab-(Cys-L-SO1861)^(3,8) or the combination ofCetuximab-(Lys-L-HSP27BNA)⁴+77 nM cetuximab did not reveal anysignificant gene silencing activity (IC50>100 nM). When the experimentwas performed in EGFR non-expressing cells (A2058) no gene silencingactivity was observed in the 1T2C combination (IC50>100 nM; FIG. 8-5D).All this shows that the 1T2C system efficiently delivers an antisenseBNA oligo to the cytoplasm of high EGFR expressing cells, therebyinducing mRNA degradation of the BNA target mRNA resulting in targetgene silencing.

Example 17

The 1 target 2-components system (1T2C) can also be the combinationtreatment of mAb1-(scaffold(-SO1861)^(n))^(n) and mAb1-protein toxin asillustrated in FIG. 15-5. Dendron(-L-SO1861)⁴ was conjugated tocetuximab via cysteine residues (Cys) conjugation with a DAR3,9 andcetuximab-Cys-(dendron(-L-SO1861)⁴)^(3,9) was tested for enhanced cellkilling activity in combination with an anti-EGFR antibody-protein toxinconjugate (cetuximab-saporin) in EGFR expressing cells (MDA-MB-468).Cetuximab-Cys-(dendron(-L-SO1861)⁴)^(3,9)+10 pM cetuximab-saporinefficiently induces toxin-mediated cell killing in high EGFR expressingcells (IC50=0.4 nM; FIG. 9-5A), whereas this was not induced bycetuximab-Cys-(dendron(-L-SO1861)⁴)^(3,9) or cetuximab+10 pMcetuximab-saporin or cetuximab (FIG. 9-5A). This shows that according tothe 1T2C invention, cetuximab conjugated dendron(-L-SO1861)⁴ efficientlyenhances endosomal escape of the cetuximab conjugated protein toxin (atnon-effective concentrations), thereby inducing cell killing of highHER2 expressing cells. Similar 1T2C experiments were performed in cellsthat express low levels of EGFR (HeLa, EGFR^(+/−)) and this revealed nocell killing activity when the 1T2C combination, according of theinvention was used (IC50>100 pM; FIG. 9-5B) indicating that in theabsence of sufficient EGFR receptor expression, effective intracellularSO1861 concentrations are not optimal (threshold) to induce cytoplasmicdelivery of the protein toxin that results in toxin-mediated cellkilling.

Next, dendron(-L-SO1861)⁴ was conjugated to the anti-HER2 antibody,trastuzumab via cysteine conjugation (Cys) with a DAR4,trastuzumab-Cys-(dendron(-L-SO1861)⁴)⁴ and tested for enhanced cellkilling activity in combination with an anti-HER2 antibody-protein toxinconjugate (trastuzumab-saporin) in HER2 expressing cells (SK-BR-3,HER2⁺⁺). Trastuzumab-Cys-(dendron(-L-SO1861)⁴)⁴+50 pMtrastuzumab-saporin efficiently induces toxin-mediated cell killing(IC50=2 nM, FIG. 9-5C), whereas this was not induced bytrastuzumab-Cys-(dendron(-L-SO1861)⁴)⁴ or trastuzumab+50 nMtrastuzumab-saporin or trastuzumab (FIG. 9-5C). This shows thattrastuzumab conjugated dendron(-L-SO1861)⁴ efficiently enhancesendosomal escape of the trastuzumab conjugated protein toxin (atnon-effective concentrations), thereby inducing cell killing of highHER2 expressing cells. Similar experiments in cells that express lowlevels of HER2 (JIMT-1, HER2^(+/−)) revealed no activity ofTrastuzumab-Cys-(dendron(-L-SO1861)⁴)⁴+50 pM trastuzumab-saporin(IC50>200 nM; FIG. 9-5D) indicating that in the absence of sufficientHER2 receptor expression, effective intracellular SO1861 concentrationsare not optimal (threshold) to induce endosomal protein toxin escape andtoxin-mediated cell killing.

Example 18

The clinical approved ADC, trastuzuzmab-emtansine (T-DM1) is a conjugateof the anti-Her2 antibody, trastuzumab and the small molecule toxinemtansine (DAR3.5). T-DM1 was titrated in combination withtrastuzumab-(Cys-L-SO1861)⁴ and compared with the antibody protein toxinconjugate, trastuzumab-saporin+Trastuzumab-(Cys-L-SO1861)⁴, according tothe invention. Whereas trastuzumab-saporin+2.5 nMtrastuzumab-(Cys-L-SO1861)⁴ showed enhanced activity compared toTrastuzumab-saporin+2.5 nM trastuzumab or trastuzumab-saporin alone(IC50=2 pM, FIG. 10-5), T-DM1+25.6 nM trastuzumab-(Cys-L-SO1861)⁴ showedno enhanced cell killing activity (IC50>100 pM; FIG. 10-5). This showsthat the 1T2C system, according to the invention, cannot enhance thedelivery of an antibody small molecule conjugate, since small moleculescan already passively cross (endolysosomal) membranes.

Example 19

FIG. 11-5A-D displays the relative cell viability when trastuzumab (FIG.11-5A), cetuximab (FIG. 11-5B) or T-DM1 (FIG. 11-5C), unconjugatedprotein toxins, saporin, dianthin and saporin conjugated to a (non-cellbinding) IgG antibody (FIG. 11-5D) are administrated to various cancercell lines SK-BR-3, JIMT-1, MDA-MB-468, A431, CaSki, HeLa, A2058.

Trastuzumab and cetuximab do not or hardly influence cell viability whenexposed to most of the cell lines, with some effect on cell growthinhibition via blocking the function of the HER2 growth factor receptorwhen trastuzumab is exposed to SK-BR-3 cells at relatively high dose andwith some effect on cell growth inhibition via blocking the function ofthe EGFR growth factor receptor when cetuximab is exposed to MDA-MB-468cells at relatively high dose.

TDM-1, or ado-trastuzumab emtansine, is a targeted therapy approved bythe U.S. Food and Drug Administration to treat: HER2-positive metastaticbreast cancer that has previously been treated with Herceptin (chemicalname: trastuzumab) and taxane chemotherapy; early-stage HER2-positivebreast cancer after surgery if residual disease was found afterneoadjuvant (before surgery) treatment with Herceptin and taxanechemotherapy. The TDM-1 is a combination of Herceptin (Trastuzumab) andthe chemotherapy medicine emtansine. FIG. 11-5C shows that the TDM-1results in decreased cell viability for all cell lines tested at >1000pM concentrations

The free toxins saporin and dianthin and the toxin saporin coupled to acontrol IgG with no affinity for any of the cell surface molecules onthe cell lines tested, do not or hardly have any influence on cellviability over a wide range of concentrations toxin tested, up to100.000 pM (FIG. 11-5D).

Example 20

The 1 target 2-components system (1T2C) can also be the combinationtreatment of mAb1-QSmix (mixture of saponins from Quillaja saponaria)and mAb1-protein toxin.

QSmix-EMCH was conjugated via cysteine residues (Cys) to cetuximab(monoclonal antibody recognizing and binding human EGFR), with a DAR 4.1(cetuximab-(Cys-L-QSmix)^(4.1)). Cetuximab-(Cys-L-QSmix)^(4.1) wastitrated on a fixed concentration of 10 pM cetuximab-saporin or 10 pMcetuximab-dianthin and targeted protein toxin mediated cell killing onA431 (EGFR⁺⁺), CaSKi (EGFR⁺) and A2058 (EGFR⁻) cells was determined.This revealed strong cell killing at low concentrations ofcetuximab-(Cys-L-QSmix)^(4.1)+10 pM cetuximab-saporin or 10 pMcetuximab-dianthin in A431 (EGFR⁺⁺) and CaSKi (EGFR⁺) cells (A431:IC50=3 nM, FIG. 12-5A; CaSKi: IC50=1 nM, FIG. 12-5B) whereas all controltreatments could not induce any cell killing in EGFR expressing cells.In cells that do not express EGFR (A2058; EGFR⁻) no HSP27 gene silencingis observed with the combination, according to the invention (IC50>1000nM; FIG. 12-5C). This shows that cetuximab conjugated QS21mixefficiently enhances endosomal escape of the cetuximab conjugatedprotein toxin (at non-effective concentrations), thereby inducing cellkilling only in EGFR expressing cells.

Example 21 2 Target 2-Component System (In Vivo)

The 2 target 2-components system (2T2C) is the combination treatment ofmAb1-SO1861 and mAb2-protein toxin, (FIG. 15-6; 16-6; 17-6). SO1861-EMCHwas conjugated via cysteine residues (Cys) to cetuximab (monoclonalantibody recognizing and binding human EGFR), with a DAR 4 resulting inthe production of: cetuximab-(Cys-L-SO1861)⁴. The combination ofcetuximab-(Cys-L-SO1861)⁴ and trastuzumab-saporin or CD71mab-saporin wastested in a A431 (EGFR⁺⁺/HER2^(+/−)/CD71⁺) xenograph ‘nude’ mouse tumormodel for EGFR tumor targeted cell killing as illustrated in FIG. 1-6and FIG. 2-6. Dose escalation was performed to determine the therapeuticefficacy (Day 9: 0.3 mg/kg trastuzumab-saporin or 0.1 mg/kgCD71mab-saporin+5 mg/kg cetuximab-(Cys-L-SO1861)⁴; Day 14, 18: 0.1 mg/kgtrastuzumab-saporin or 0.05 mg/kg CD71mab-saporin+5 mg/kgcetuximab-(Cys-L-SO1861)⁴; Day 21: 0.05 mg/kg trastuzumab-saporin or0.05 mg/kg CD71mab-saporin+15 mg/kg cetuximab-(Cys-L-SO1861)⁴; Day 28:0.02 mg/kg trastuzumab-saporin or 0.02 mg/kg CD71mab-saporin+15 mg/kgcetuximab-(Cys-L-SO1861)⁴ trastuzumab-saporin/cetuximab-SO1861. Controlswere on the same dosing scheme respectively, only cetuximab (i. v.) wasgiven 25 mg/kg every treatment day). At day 32 (dashed line), 35 and 39we started the combination, according to the 2T2C invention of 25 mg/kgcetuximab-(Cys-L-SO1861)⁴ (intraperitoneal injection (i.p.)+0.02 mg/kgtrastuzumab-saporin or 0.02 CD71mab-saporin (intravenous administration,(i.v.)) and this revealed strong tumor regression for both 2T2Ccombination groups compared to the vehicle control, 25 mg/kgcetuximab-(Cys-L-SO1861)⁴ or 0.02 mg/kgtrastuzumab-saporin/CD71mab-saporin mono therapies (FIG. 1-6, 2-6). The2T2C system even outcompetes cetuximab, the clinically used monoclonalantibody against EGFR. Next we performed the same experiment but then westarted with 25 mg/kg cetuximab-(Cys-L-SO1861)⁴ (intraperitonealinjection (i.p.)+0.03 mg/kg trastuzumab-saporin or 0.03 CD71mab-saporin(intravenous administration, (i.v.)) treatment with a dosing at day 9and 14 and thereafter 1 dosing per week. The 2T2C system according tothe invention showed tumor regression in all mice and even in 1 mice inboth 2T2C groups, complete tumor eradication (tumor volume=0 mm³) (FIG.2-6). Also here the controls showed a strong increased in tumor volumewhereas the positive control for this A431 mice model, cetuximab showedonly tumor growth inhibition, but no regression (FIG. 2-6). This showsand enables the 2T2C system approach, according to the invention, ofcetuximab conjugated SO1861+trastuzumab conjugated protein toxin orCD71mab conjugated protein toxin inducing highly efficient targeteddelivery of a therapeutic protein toxin in the cytoplasm of solid tumorsof tumor bearing mice, in vivo, thereby inducing even full tumoreradication in some mice and strong tumor regression in others even inlarge size tumors (2000 mm³).

Example 22 2 Target 2-Component System (In Vitro) Results

The 2 target 2-components system (2T2C) is the combination treatment ofmAb1-SO1861 and mAb2-protein toxin, (see also FIG. 1-6, 2-6, 15-6, 16-6,17-6). SO1861-EMCH was conjugated via cysteine residues (Cys) tocetuximab (monoclonal antibody recognizing and binding human EGFR), witha DAR 3,7 (cetuximab-(Cys-L-SO1861)^(3,7)).Cetuximab-(Cys-L-SO1861)^(3,7) was titrated on a fixed concentration of50 pM trastuzumab-saporin (trastuzumab, conjugated to the protein toxin,saporin) and targeted protein toxin mediated cell killing on EGFR/HER2expressing cells (A431, EGFR⁺⁺/HER2^(+/−); CaSKi, EGFR⁺E/HER2^(+/−)) wasdetermined as illustrated in FIG. 3-6. This revealed strong cell killingat low concentrations of cetuximab-(Cys-L-SO1861)^(3,7) (A431: IC50=3 nMand CaSKi IC50=10 nM; FIG. 3-6A, 3-6B) whereas equivalent concentrationscetuximab, cetuximab-(Cys-L-SO1861)^(3,7) or cetuximab+50 pMtrastuzumab-saporin could not induce any cell killing activity inEGFR/HER2 expressing cells. This shows that relatively lowconcentrations of cetuximab-SO1861 conjugate efficiently enhancesendosomal escape of the trastuzumab conjugated protein toxin (atnon-effective concentrations), thereby inducing efficient cell killingof high EGFR/low HER2 expressing cells.

Next, trastuzumab-saporin was titrated on a fixed concentration of 75 nMcetuximab-(Cys-L-SO1861)^(3,7) and targeted protein toxin mediated cellkilling on EGFR/HER2 expressing cells was determined. This revealed that75 nM cetuximab-(Cys-L-SO1861)^(3,7) in combination with lowconcentrations trastuzumab-saporin induced already efficient cellkilling in EGFR/HER2 expressing cells (A431: IC50=5 pM; and CaSKi:IC50=1 pM; FIGS. 3-6C and 3-6D), whereas trastuzumab-saporin alone ortrastuzumab-saporin+75 nM cetuximab did not show significant cellkilling activity (IC50>10.000 pM) in both cell lines (FIG. 3-6C, 3-6D).All this shows that relatively low concentrations of trastuzumab-saporincan be effective and induce cell killing in combination with lowcetuximab-SO1861 conjugate concentrations in high EGFR/low HER2expressing cells.

Next, cetuximab-(Cys-L-SO1861)^(3,7) was titrated on a fixedconcentration of 50 pM trastuzumab-saporin and targeted proteintoxin-mediated cell killing on HeLa (EGFR^(+/−)/HER2^(+/−)) or A2058(EGFR/HER2^(+/−)) was determined as illustrated in FIG. 4-6, 15-6=17-6.Both HeLa (EGFR^(+/−)/HER2^(+/−)) and A2058 (EGFR/HER2^(+/−)) cells donot show cell killing at low concentrations ofcetuximab-(Cys-L-SO1861)^(3,7)+50 pM trastuzumab-saporin (HeLa: IC50=400nM; A2058: IC50>400 nM; FIG. 4-6A, 4-6B). This shows that in the absenceof sufficient receptor expression, effective intracellular deliveredSO1861 concentrations are not reached (threshold) to induce endosomalescape and cytoplasmic delivery of the protein toxin. Next,trastuzumab-saporin was titrated on a fixed concentration of 75 nMcetuximab-(Cys-L-SO1861)³⁷ and targeted protein toxin mediated cellkilling on HeLa (EGFR^(+/−)/HER2^(+/−)) or A2058 (EGFR/HER2^(+/−)) wasdetermined. Both HeLa (EGFR^(+/−)/HER2^(+/−)) and A2058(EGFR/HER2^(+/−)) cells showed no cell killing activity (HeLa:IC50>10.000 pM; A2058: IC50>10.000 pM; FIG. 4-6C, 4-6D). All this showsthat cells with low or no EGFR receptor expression are not susceptiblefor the combination ofcetuximab-(Cys-L-SO1861)^(3,7)+trastuzumab-saporin, due to a lack ofsufficient EGFR receptor that facilitates the antibody-mediated deliveryof sufficient SO1861 (threshold) to ensure endosomal escape of the toxinwithin the cytoplasm of the cell.

Next, SO1861-EMCH was conjugated via cysteine residues (Cys) totrastuzumab (monoclonal antibody recognizing and binding human HER2),with a DAR 4 (trastuzumab-(Cys-L-SO1861)⁴). Trastuzumab-(Cys-L-SO1861)⁴was titrated on a fixed concentration of 1.5 pM EGFdianthin (EGFRtargeted ligand toxin fusion protein) and targeted protein toxinmediated cell killing on HER2/EGFR expressing cells (SK-BR-3:HER2⁺⁺/EGFR^(+/−)) was determined. This revealed strong cell killing atlow concentrations of trastuzumab-(Cys-L-SO1861)⁴+1.5 pM EGFdianthin(SK-BR-3: IC50=1 nM; FIG. 5-6A) whereas equivalent concentrationstrastuzumab, trastuzumab-(Cys-L-SO1861)⁴ or trastuzumab+1.5 pMEGFdianthin could not induce any cell killing activity inHER2⁺⁺/EGFR^(+/−) expressing cells. This shows that trastuzumabconjugated SO1861 efficiently enhances endosomal escape of the EGFfusion protein toxin (at non-effective concentrations), thereby inducingcell killing of high HER2/low EGFR expressing cells.

Next, EGFdianthin was titrated on a fixed concentration of 2.5 nMtrastuzumab-(Cys-L-SO1861)⁴ and targeted protein toxin mediated cellkilling on SK-BR-3 (HER2⁺⁺/EGFR^(+/−)) expressing cells was determined.This revealed that 2.5 nM trastuzumab-(Cys-L-SO1861)⁴ in combinationwith low concentrations EGFdianthin induced already efficient cellkilling in HER2/EGFR expressing cells (SK-BR-3: IC50=1 pM) (FIG. 5-6B),whereas EGFdianthin alone or EGFdianthin+2.5 nM trastuzumab showed nocell killing activity (IC50>10.000 pM) (FIG. 5-6B). All this shows thatrelatively low concentrations of EGFdianthin can be effective and inducecell killing only in combination with low trastuzumab-(Cys-L-SO1861)⁴concentrations in high HER2/low EGFR expressing cells.

Next, trastuzumab-(Cys-L-SO1861)⁴ was titrated on a fixed concentrationof 1.5 pM EGFdianthin and targeted protein toxin mediated cell killingon JIMT-1 (HER2^(+/−)/EGFR^(+/−)) or MDA-MB-468: HER2⁻/EGFR⁺⁺) wasdetermined. Both cell lines were not sensitive for any combination oftrastuzumab-(Cys-L-SO1861)⁴+1.5 pM EGFdianthin (JIMT-1: IC50>1000 nM;MDA-MB-468: IC50>1000 nM; FIG. 6-6A, 6-6B). This shows that in theabsence of sufficient HER2 receptor expression, effective intracellulardelivered SO1861 concentrations are not reached (threshold) to induceendosomal escape and cytoplasmic delivery of the protein toxin.

Next, EGFdianthin was titrated on a fixed concentration of 2.5 nMtrastuzumab-(Cys-L-SO1861)⁴ and targeted protein toxin mediated cellkilling on JIMT-1 (HER2^(+/−)/EGFR^(+/−)) or MDA-MB-468 (HER2⁻/EGFR⁺⁺)was determined. Both cell lines showed cell killing at high EGFdianthinconcentrations with or without 2.5 nMtrastuzumab-(Cys-L-SO1861)⁴(JIMT-1: IC50=10.000 pM; MDA-MB-468: IC50=200pM FIG. 6-6C, 6-6D).

All this shows that cells with low or no HER2 receptor expression arenot susceptible for the combination oftrastuzumab-(Cys-L-SO1861)^(3,7)+1.5 pM EGFdianthin, due to a lack ofsufficient HER2 receptor that facilitates the antibody-mediated deliveryof sufficient SO1861 (threshold) to ensure endosomal escape of the toxinwithin the cytoplasm of the cell.

Next, SO1861-EMCH was conjugated via cysteine residues (Cys) totrastuzumab (monoclonal antibody recognizing and binding human HER2),with a DAR 4, (trastuzumab-(Cys-L-SO1861)⁴). Trastuzumab-(Cys-L-SO1861)⁴was titrated on a fixed concentration of 5 pM cetuximab-saporin (EGFRtargeting antibody-protein toxin conjugate) and targeted protein toxinmediated cell killing on HER2/EGFR expressing cells (SK-BR-3:HER2⁺⁺/EGFR^(+/−)) was determined as illustrated in FIG. 15-6-17-6. Thisrevealed strong cell killing at low concentrations oftrastuzumab-(Cys-L-SO1861)⁴+5 pM cetuximab-saporin (SK-BR-3: IC50=1 nM;FIG. 7-6A) whereas equivalent concentrations trastuzumab,trastuzumab-(Cys-L-SO1861)⁴ or trastuzumab+5 pM cetuximab-saporin couldnot induce any cell killing activity in HER2⁺⁺/EGFR^(+/−) expressingcells. This shows that trastuzumab conjugated SO1861 efficientlyenhances endosomal escape of the cetuximab conjugated protein toxin (atnon-effective concentrations), thereby inducing cell killing ofHER2⁺⁺/EGFR^(+/−) expressing cells.

Next, cetuximab-saporin was titrated on a fixed concentration of 2.5 nMtrastuzumab-(Cys-L-SO1861)⁴ and 75 nM trastuzumab-(Cys-L-SO1861)⁴ andtargeted protein toxin mediated cell killing on HER2/EGFR expressingcells (SK-BR-3: HER2⁺⁺/EGFR^(+/−)) was determined. This revealed that2.5 nM trastuzumab-(Cys-L-SO1861)⁴ in combination with lowconcentrations cetuximab-saporin induced already efficient cell killingin SK-BR-3 cells (SK-BR-3: IC50=1 pM; FIG. 7-6B), whereascetuximab-saporin alone or cetuximab-saporin+2.5 nM trastuzumab showedcell killing only at high concentrations trastuzumab-saporin (SK-BR-3:IC50>4000 pM; FIG. 7-6B). All this shows that relatively lowconcentrations of cetuximab-saporin can be effective and induce cellkilling only in combination with low trastuzumab-(Cys-L-SO1861)⁴concentrations in HER2⁺⁺/EGFR^(+/−) expressing cells.

Next, trastuzumab-(Cys-L-SO1861)⁴ was titrated on a fixed concentrationof 5 pM cetuximab-saporin and targeted protein toxin mediated cellkilling on JIMT-1 (HER2^(+/−)/EGFR^(+/−)) and MDA-MB-468 (HER2⁻/EGFR⁺⁺)cells was determined. Both cell lines were not sensitive for thecombination of trastuzumab-(Cys-L-SO1861)⁴+5 pM cetuximab-saporin(JIMT-1: IC50>1000 nM; MDA-MB-468: IC50>1000 nM; FIG. 8-6A, 8-6B). Thisshows that in the absence of sufficient HER2 receptor expression,effective intracellular delivered SO1861 concentrations are not reached(threshold) to induce endosomal escape and cytoplasmic delivery of theprotein toxin.

Next, cetuximab-saporin was titrated on a fixed concentration of 2.5 nMtrastuzumab-(Cys-L-SO1861)⁴ and targeted protein toxin mediated cellkilling on JIMT-1 (HER2^(+/−)/EGFR^(+/−)) and MDA-MB-468 (HER2⁻/EGFR⁺⁺)cells was determined. Both cell lines showed cell killing at similarcetuximab-saporin concentrations with or without 2.5 nMtrastuzumab-(Cys-L-SO1861)⁴(JIMT-1: IC50=80 pM; MDA-MB-468: IC50=100 pM;FIG. 8-6C, 8-6D).

All this shows that cells with low or no HER2 receptor expression arenot susceptible for the combination oftrastuzumab-(Cys-L-SO1861)⁴+cetuximab-saporin, due to a lack ofsufficient HER2 receptor that facilitates the antibody-mediated deliveryof sufficient SO1861 (threshold) to ensure endosomal escape of the toxinwithin the cytoplasm of the cell.

Example 23

In order to show that the activity of the conjugated SO1861 is driven bythe acidification of the endosomal compartments, the 2T2 componentssystem, according to the invention was tested in combination with anendosomal acidification inhibitor, chloroquine. Trastuzumab-saporin+77nM cetuximab-(Cys-L-SO1861)^(3,9) or trastuzumab-dianthin+77 nMcetuximab-(Cys-L-SO1861)^(3,9) showed strong cell killing activity inA431 (EGFR⁺⁺/HER2^(+/−)) cells, whereas this 2T2C activity, according tothe invention, was inhibited when 800 nM chloroquine wasco-administrated to both combinations (FIG. 9-6A). Same results wereobserved when CD71mab-saporin+10.5 nM cetuximab-(Cys-L-SO1861)^(3,9)+500nM chloroquine was tested in A431 (EGFR⁺⁺/CD71⁺) and MDA-MB-468(EGFR⁺⁺/CD71⁺) cells (FIG. 9-6B, 9C) or when CD71mab-saporin+5 nMtrastuzumab-(Cys-L-SO1861)⁴+500 nM chloroquine was tested in SK-BR-3(HER2⁺⁺/CD71⁺) cells (FIG. 9-6D). This shows that the intracellularactivity of conjugated SO1861 within the 2T2C system can be inhibitedwhen acidification of endosomes is blocked.

Example 24

The 2 target 2-components system (2T2C) is also the combinationtreatment of mAb1-SO1861 and mAb2-antisense BNA oligo nucleotide, (FIG.16-6). Therefore, the 2T2C system was also tested in combination with anantisense BNA oligonucleotide against the mRNA of a cancer specifictarget gene, heat shock protein 27 (HSP27). Upon release into thecytoplasm the antisense BNA recognizes and binds the mRNA encoding forHSP27, targeting the mRNA for destruction thereby depleting the HSP27expression within the cancer cell. HSP27BNA was conjugated totrastuzumab with a DAR4.4 (trastuzumab-(Lys-L-HSP27BNA)⁴′⁴) and testedin combination with Cetuximab-(Cys-L-SO1861)^(3,9) for enhanced HSP27gene silencing activity in A431 (EGFR⁺⁺/HER2^(+/−)) cells and A2058(EGFR/HER2^(+/−)) cells as illustrated in FIG. 16-6.Cetuximab-(Cys-L-SO1861)³′⁹ was titrated on a fixed concentration of 100nM Trastuzumab-(Lys-L-HSP27BNA)^(4,4) and targeted HSP27BNA-mediatedgene silencing activity was determined.Cetuximab-(Cys-L-SO1861)^(3,9)+100 nM Trastuzumab-(Lys-L-HSP27BNA)^(4,4)show strong gene silencing activity in A431 cells (EGFR⁺⁺/HER2^(+/−))(A431: IC50=1 nM; FIG. 10-6A), compared toCetuximab-(Cys-L-SO1861)^(3,9) alone. In A2058 cells (EGFR⁻/HER2^(+/−)),the combination according to the invention showed no HSP27 genesilencing (A2058: IC50>100 nM; FIG. 10-6B). This shows that cetuximabconjugated SO1861 efficiently enhances endosomal escape of thetrastuzumab conjugated BNA oligo nucleotide (at non-effectiveconcentrations), thereby inducing target gene silencing inEGFR⁺⁺/HER2^(+/−) expressing cells.

Next, Trastuzumab-(Lys-L-HSP27BNA)^(4,4) was titrated on a fixedconcentration of Cetuximab-(Cys-L-SO1861)^(3,9) and targetedHSP27BNA-mediated gene silencing activity was determined in A431(EGFR⁺⁺/HER2^(+/−)) cells and A2058 (EGFR/HER2^(+/−)) cells asillustrated in FIG. 16-6. Trastuzumab-(Lys-L-HSP27BNA)^(4,4)+77 nMCetuximab-(Cys-L-SO1861)^(3,9) show strong gene silencing activity inA431 cells (EGFR⁺⁺/HER2^(+/−)) (A431: IC50=1 nM; FIG. 10-6C), whereastrastuzumab-(Lys-L-HSP27BNA)^(4,4) alone orCetuximab-(Cys-L-SO1861)^(3,9) alone ortrastuzumab-(Lys-L-HSP27BNA)^(4,4)+77 nM cetuximab did not reveal anysignificant gene silencing activity (IC50>100 nM). A2058(EGFR⁻/HER2^(+/−)) cells did not show any gene silencing activity in thecombination according to the invention (A2058: IC50>100 nM; FIG. 10-6D).All this shows that relatively low concentrations oftrastuzumab-HSP27BNA can be effective and induce cell killing only incombination with low concentrations of cetuximab-(-L-SO1861)concentrations in HER2⁺⁺/EGFR^(+/−) expressing cells.

Example 25

The 2 target 2-components system (2T2C) can also be the combinationtreatment of mAb1-(dendron(-SO1861)^(n))^(n) and mAb2-protein toxin.Dendron(-L-SO1861)⁴ was conjugated to the anti-EGFR antibody, cetuximabvia cysteine residues (Cys) with a DAR3,9,(cetuximab-Cys-(dendron(-L-SO1861)⁴)^(3,9)) and tested for enhanced cellkilling activity in combination with an anti-CD71 antibody protein toxinconjugate (CD71mab-saporin) in MDA-MB-468 (EGFR⁺⁺/CD71⁺) expressingcells as illustrated in FIG. 17-6.Cetuximab-Cys-(dendron(-L-SO1861)⁴)^(3,9)+10 pM CD71mab-saporinefficiently induces toxin-mediated cell killing in MDA-MB-468(EGFR⁺⁺/CD71⁺) expressing cells (IC50=0.4 nM, FIG. 11-6A), whereas thiscould not be induced by Cetuximab-Cys-(dendron(-L-SO1861)⁴)^(3,9)) orcetuximab+10 pM CD71mab-saporin or cetuximab (FIG. 11-6A). This showsthat cetuximab conjugated dendron(-L-SO1861)⁴ efficiently enhancesendosomal escape of the CD71mab-protein toxin (at non-effectiveconcentrations), thereby inducing cell killing of EGFR⁺⁺/CD71⁺expressing cells. Similar experiments were performed in HeLa cells(HER2^(+/−)/CD71⁺) cells and this revealed no activity ofcetuximab-Cys-(dendron(-L-SO1861)⁴)^(3,9))+10 pM CD71mab-saporin(IC50>100 nM FIG. 11-6B) indicating that in the absence of sufficientEGFR receptor expression, effective intracellular SO1861 concentrationsare not reached (threshold) to induce endosomal escape and cytoplasmicdelivery of the protein toxin.

Next, dendron(-L-SO1861)⁴ was conjugated to the anti-HER2 antibody,trastuzumab via cysteine conjugation (Cys) with a DAR4,trastuzumab-Cys-(dendron(-L-SO1861)⁴)⁴ and tested for enhanced cellkilling activity in combination with an anti-CD71 antibody protein toxinconjugate (CD71mab-saporin) in SK-BR-3 cells (HER2⁺⁺/CD71⁺) expressingcells. Trastuzumab-Cys-(dendron(-L-SO1861)⁴)⁴+10 pM CD71mab-saporinefficiently induces toxin-mediated cell killing in SK-BR3 cells (IC50=3nM, FIG. 11-6C), whereas this was not induced bytrastuzumab-Cys-(dendron(-L-SO1861)⁴)⁴ or trastuzumab (equivalent)+10 pMCD71mab-saporin or trastuzumab (FIG. 11-6C). This shows that trastuzumabconjugated dendron(-L-SO1861)⁴, according to the invention efficientlyenhances endosomal escape of the CD71mab-protein toxin (at non-effectiveconcentrations), thereby inducing cell killing of HER2⁺⁺/CD71⁺expressing cells. Similar experiments were performed in JIMT-1 cells(HER2^(+/−)/CD71⁺) and this revealed no activity oftrastuzumab-Cys-(dendron(-L-SO1861)⁴)⁴+10 pM CD71mab-saporin (IC50>100nM FIG. 11-6C) indicating that in the absence of sufficient HER2receptor expression, effective intracellular SO1861 concentrations arenot reached (threshold) to induce endosomal escape and cytoplasmicdelivery of the protein toxin.

Example 26

The clinical approved ADC, trastuzumab-emtansine (T-DM1) is a conjugateof the anti-Her2 antibody, trastuzumab and the small molecule toxinemtansine (DAR3-4). T-DM1 was tested within the 2T2C system, accordingto the invention in combination with cetuximab-(Cys-L-SO1861)⁴. T-DM1+77nM cetuximab-(Cys-L-SO1861)^(3,9) showed no enhanced cell killingactivity compared to T-DM1 alone or T-DM1+77 nM cetuximab (IC50=80.000pM, FIG. 12-6), whereas trastuzumab-saporin+75 nMcetuximab-(Cys-L-SO1861)^(3,7), according to the invention showedenhanced cell killing activity compared to trastuzumab-saporin+75 nMcetuximab or trastuzumab-saporin alone (IC50=3 pM, FIG. 12-6). All thisshows that the 2T2C system does not enhance the delivery of antibodyconjugated small molecules, that are already able to passively crosscellular (endosomal) membranes.

Example 1-2

Various concentrations of trastuzumab-saporin (HER2 targetedprotein-toxin conjugate; intravenous) were tested in combination with1.5 mg/kg SO1861 (1 hour before antibody-toxin injection; subcutaneous)for enhanced efficacy in a BT474 (HER2++) xenograph mouse model. Dosingstarted at day 13 when tumors reached ˜150 mm³ in size and tumor volumewas determined after every treatment. Although tumor growth inhibitionwas observed in the mice treated with 1 mg/kg and 0.3 mg/kgtrastuzumab-saporin, there was no enhanced tumor growth inhibitionobserved in the mice treated with the combination oftrastuzumab-saporin+SO1861. This shows that unconjugated SO1861 is notable to enhance antibody-protein toxins within the current settings andmouse model.

Example 2-2 Materials:

QSmix (1): S4521 (Sigma Aldrich); QSmix (2): 6857.1 (Carl Roth) QSmix(3): Quil-A® Adjuvant: vac-quil (InvivoGen/Brenntag).

Previously, the efficacy of various saponins (SO1861, SO1642) were coadministrated as ‘free’ unconjughated molecules to cells in combinationwith a ligand toxin fusion (e.g. EGFdianthin) or an antibody-proteintoxin conjugate, resulting in enhanced cell killing activity of targetexpressing cells. Here, three different saponin molecules (SO1861,SO1862 (isomer of SO1861), SO1832 and SO1904) isolated from a rootextract of Saponaria officinalis were titrated in the presence andabsence of a non-effective fixed concentration of 1.5 pM EGFdianthin onHeLa (EGFR⁺) cells. This revealed a strong enhancement of cell killingactivity for all tested saponin variants (IC50=300 nM; FIG. 2-2A)compared to the treatments without EGFdianthin. Next, EGFdianthin wastitrated with a fixed concentration of saponin (˜1000 nM) and thisrevealed strong targeted cell killing enhancement at low pMconcentrations of EGFdianthin (IC50=0.4 pM; FIG. 2-2B), observed for allused saponins SO1861, SO1862 (isomer of SO1861), SO1832 and SO1904.EGF-dianthin alone could only induce cell killing at very highconcnetrations (IC50=10.000 pM). This shows that these specific types ofsaponins, all have the intrinsic capacity to efficiently induceendosomal escape with only a very low amount of targeted toxinavailable.

To extend this test, saponins from other sources were analyzed. Asaponin purified from a root extract of Gypsophila elegans M. Bieb.(GE1741) was titrated on HeLa cells in the presence and absence of 1.5pM EGFdianthin and compared with purified SO1861. GE1741 also enhancesthe EGFdianthin induced HeLa cell killing, but shows slightly lessefficacy compared to SO1861. (GE1741 IC50=800 nM; FIG. 2-2C) and alsodisplays a higher general toxicity (IC50=5.000 nM in absence ofEGFdianthin; FIG. 2-2C). A similar test in which different partiallypurified mixtures of Quillaja saponaria saponins (QSmix 1-3) wereco-administrated with 1.5 pM EGFdianthin on HeLa cells and this revealedfor 2 out of 3 (QSmix 1 and QSmix 3) similar activity as SO1861 (IC50QSmix/QSmix3=300 nM; FIG. 2-2D). QSmix (2) is less efficient inenhancing 1.5 pM EGFdianthin induced cell killing (IC50=2000 nM; FIG.2-2D), however, no general toxicity is observed. This shows that also inQS extracts, specific type of saponins are available that efficientlyinduce endosomal escape of the targeting ligand toxin EGFdianthin.

Example 3-2

In order to conjugate SO1861 molecules to antibodies, according to theinvention, labile/acid sensitive linkers (-EMCH or -N3), was conjugatedto SO1861 via the aldehyde group, producing SO1861-EMCH or SO1861-N3. Toverify the activity of SO1861-EMCH the molecule was titrated in thepresence and absence of a fixed non-effective (1.5 pM) EGFdianthinconcentration on EGFR expressing (A431, HeLa) and non-expressing cells(A2058). In all three cell lines SO1861 alone showed a strong cellviability reduction, whereas SO1861-EMCH as single compound showed notoxicity up to 25.000 nM (FIG. 3-2A-C). When SO1861-EMCH was combinedwith 1.5 pM EGFdianthin a strong target specific cell viabilityreduction is observed in the EGFR+A431 and HeLa cells (IC50=3.000 nM;FIG. 3-2A,B), while the EGFR⁻ A2058 cells are not affected at all (FIG.3-2C). Similar results were obtained for SO1861-N3. 501861-N3co-administrated with 1.5 pM EGFdianthin also shows efficient cellkilling on A431 and HeLa cells (IC50=3.000 nM), but without EGFdianthina general toxicity is observed at above 10.000 nM (FIG. 3-2D, 3-2E).

For the stable conjugation of SO1861 to antibodies, according to theinvention, a stable linker (HATU) was conjugated to SO1861 via thecarboxylic acid group of SO1861 producing, SO1861-(S). To determine theactivity different concentrations of SO1861-(S) were co-administratedwith 1.5 pM EGFdianthin and tested for cell killing activity in EGFRexpressing HeLa cells. SO1861-(S) showed a similar activity as SO1861,indicating that conjugation to the carboxylic acid does not affect theendosomal escape enhancing potency of the molecule as is observed withSO1861-EMCH (FIG. 4-2).

Example 4-2

Labile SO1861 was conjugated via cysteine residues (Cys) to theanti-EGFR antibody cetuximab (monoclonal antibody recognizing andbinding human EGFR), with DAR3.9 (cetuximab-(Cys-L-SO1861)³⁹) and testedfor its enhanced delivery of antisense BNA oligo nucleotides resultingin enhanced target gene silencing. In this study we used an antisenseBNA oligonucleotide against the mRNA of a cancer specific target gene,heat shock protein 27 (HSP27). Within the cytoplasm of the cell HSP27BNAbind the mRNA encoding for HSP27, target the mRNA for destruction,thereby reducing the HSP27 expression within the cancer cell.Cetuximab-(Cys-L-SO1861)³⁹ was titrated on fixed concentration of 100 nMHSP27BNA on EGFR⁺⁺ (A431) and EGFR⁻ (A2058) cells. The combinationaccording of the invention showed efficient HSP27 silencing on A431(IC50=2 nM; FIG. 5-2A), while no silencing was observed forcetuximab-(Cys-L-SO1861)³⁹ alone. Cetuximab-(Cys-L-SO1861)³⁹+100 nMHSP27BNA showed no gene silencing activity in EGFR⁻ cells (A2058) (FIG.5-2B). This shows that low concentrations of antibody-conjugated SO1861efficiently can enhance cytoplasmic delivery and endolysosomal escape ofan antisense BNA oligo nucleotide, thereby inducing efficient genesilencing in target expressing cells.

Next the HSP27BNA was titrated on EGFR⁺⁺ (A431) and EGFR⁻ (A2058) cellscombined with fixed concentration of cetuximab-(Cys-L-SO1861)³⁹. Thisshows that HSP27BNA in combination with 28.6 nMcetuximab-(Cys-L-SO1861)^(3.9) or 77 nM cetuximab-(Cys-L-SO1861)^(3.9)very efficiently enhances HSP27 gene silencing in A431 cells (IC50=10nM; FIG. 5-2C). HSP27BNA alone or combined with a fixed equivalent of 77nM cetuximab are less efficient (IC50=1.000 nM; FIG. 5-2C). Thecombination treatment of HSP27BNA+77 nM cetuximab-(Cys-L-SO1861)³⁹ wasalso tested on EGFR− cells (A2058) and this revealed no HSP27 genesilencing enhancement (IC50=1.000 nM; FIG. 5-2D). This shows that cellswith low or no EGFR receptor expression are not susceptible for thecombination of cetuximab-(Cys-L-SO1861)^(3,9)+HSP27BNA, while cetuximabtargeted SO1861 can enhance HSP27 gene silencing efficiently at lowconcentrations of non-targeted HS27BNA in high EGFR cells.

Next, SO1861-EMCH was conjugated via cysteine residues (Cys) tocetuximab (monoclonal antibody recognizing and binding human EGFR), witha DAR 3,8. The combination according to the invention,cetuximab-(Cys-L-SO1861)^(3,8)+HSP27BNA (antisense HSP27BNA oligonucleotide targeting and inducing degradation of the onco-target hsp27mRNA (gene silencing) in cancer cells) was tested in a A431 xenograph‘nude’ mouse tumor model for EGFR-mediated tumor targeted HSP27 genesilencing. Dosing started at day 12 when tumors reached ˜150 mm³ in sizeand HSP27 mRNA expression was determined. For this, tumor samples werecollected at 72h after the first dosing and analysed for HSP27 geneexpression levels compared to cellular control mRNA expression levels(reference genes). Tumor bearing mice (n=3) were treated(intraperitoneal; i.p.) at day 12: 25 mg/kgcetuximab-(Cys-L-SO1861)^(3,8)+25 mg HSP27BNA and at day 15: 25 mg/kgcetuximab-(Cys-L-SO1861)^(3,8)+10 mg HSP27BNA and this revealed a 25%reduction in HSP27 mRNA expression in the tumors compared to vehiclecontrol or single dosing of 25 mg/kg HSP27BNA (FIG. 6-2). This shows andenables that conjugation of SO1861 to a targeting antibody, according tothe invention, efficiently induces SO1861-mediated enhanced cytoplasmicdelivery of a therapeutic antisense oligo nucleotide in solid tumors oftumor bearing mice, inducing tumor targeted gene silencing, in vivo.

Example 1-7—Targeted Antisense Oligonucleotide Coupled to an AntibodyCetuximab-(Lys-L-HSP27BNA)+Saponin SO1861

An antisense BNA oligonucleotide against the mRNA of a cancer specifictarget gene, heat shock protein 27 (HSP27), will upon release into thecytoplasm recognize and bind the mRNA encoding for HSP27, target themRNA for destruction and thereby lower the HSP27 expression within thecancer cell. Non-targeted HSP27BNA was titrated on EGFR⁺⁺ (A431) andEGFR⁻ (A2058) cells to test for HSP27 gene silencing in combination withsaponins. Data revealed efficient HS27 silencing on both A431 A2058cells when HSP27BNA was combined with 4000 nM SO1861-ECMH (IC50=10 nM;FIG. 1-7A, 1-7B), while no silencing was observed for single HSP27BNAtreatment (IC50 1.000 nM; FIG. 1-7A, 1-7B).

Next, HSP27BNA was conjugated to the lysines of cetuximab with DAR1.5and DAR3.9, resulting in cetuximab-(Lys-L-HSP27BNA)¹⁵ andcetuximab-(Lys-L-HSP27BNA)³⁹. The conjugated cetuximab-HSP27BNA sampleswere again titrated on EGFR⁺⁺ (A431) and EGFR⁻ (A2058) cells to test fortargeted HSP27 gene silencing in combination with saponins. Theseconjugates show very efficient HSP27 gene silencing in A431 (EGFR⁺⁺)cells in the presence of 4000 nM SO1861-EMCH (DAR1.5 IC50=0.05 nM andDAR3.9 IC50=0.3 nM; FIG. 1-7A), while the silencing of the targetedHSP27BNA samples is comparable to the non-targeted HSP27BNA in theabsence of SO1861-EMCH. The silencing in A2058 (EGFR⁻) cells is notimproved compared to non-targeted HSP27BNA in general. Both in thepresence and absence of SO1861 similar HSP27BNA (conjugate) amounts arerequired to induce silencing (FIG. 1-7B). This shows that cells withhigh EGFR receptor expression very efficient targeted HSP27 genesilencing can be achieved using targeted HSP27BNA in combination withSO1861.

An antisense BNA oligonucleotide against the mRNA of a cancer specifictarget gene, heat shock protein 27 (HSP27), will upon release into thecytoplasm recognize and bind the mRNA encoding for HSP27, target themRNA for destruction and thereby lower the HSP27 expression within thecancer cell. Non-targeted HSP27BNA was titrated on EGFR⁺⁺ (A431) andEGFR⁻ (A2058) cells to test for HSP27 gene silencing in combination withsaponins. Data revealed efficient HS27 silencing on both A431 A2058cells when HSP27BNA was combined with 4000 nM SO1861-ECMH (IC50=10 nM;FIG. 1-7C, 1-7D), while no silencing was observed for single HSP27BNAtreatment (IC50 1.000 nM; FIG. 1-7C, 1-7D).

Next, HSP27BNA was conjugated to the lysines of cetuximab with DAR1.5and DAR3.9, resulting in cetuximab-(Lys-L-HSP27BNA)^(1.5) andcetuximab-(Lys-L-HSP27BNA)^(3.9). The conjugated cetuximab-HSP27BNAsamples were again titrated on EGFR⁺⁺ (A431) and EGFR⁻ (A2058) cells totest for targeted HSP27 gene silencing in combination with saponins.These conjugates show very efficient HSP27 gene silencing in A431(EGFR⁺⁺) cells in the presence of 4000 nM SO1861-EMCH, requiring lessHSP27BNA oligo (IC50=0.04 nM; FIG. 1-7C), while the silencing of thetargeted HSP27BNA samples is comparable to the non-targeted HSP27BNA inthe absence of SO1861-EMCH. The silencing in A2058 (EGFR⁻) cells is notimproved compared to non-targeted HSP27BNA in general. It even seemsthat about 10× more HSP27BNA oligo is required in the presence ofSO1861, while no significant silencing is observed without SO1861 (FIG.1-7D). This shows that cells with high EGFR receptor expression veryefficient targeted HSP27 gene silencing can be achieved using targetedHSP27BNA in combination with SO1861.

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1. Therapeutic molecule with chemical structure of COMPOUND I:A1_(m)((-L9_(w))((-L1_(q)-B1_(n))_(u)((-L2_(r)-L3_(s))(-L4_(v)-C)_(p))_(t)))_(x)  (compound I), wherein A1 is a first ligand if B1 is a first effectormoiety, or A1 is the first effector moiety if B1 is the first ligand; Cis a saponin; m=0 or 1 if A1 is the first ligand and B1 is the firsteffector moiety; m=0-32 if A1 is the first effector moiety and B1 is thefirst ligand; n=0 or 1 if B1 is the first ligand and A1 is the firsteffector moiety, or if A1 is the first ligand and B1 is the firsteffector moiety; p=any of 1-128; L1 is at least one linker forcovalently coupling two chemical groups; L2 is at least one linker forcovalently coupling two chemical groups; L3 is at least one oligomericor polymeric scaffold for covalently coupling two chemical groups; L4 isat least one linker for covalently coupling two chemical groups; L9 is atri-functional linker for covalently coupling three chemical groups; q=0or 1; r=0 or 1; s=0 or 1; t=0, 1 or 2 if s=0, and t=any of 0-16 if s=1;u=any of 0-32 if A1 is the first ligand and B1 is the first effectormoiety, or u=1 if A1 is the first effector moiety and B1 is the firstligand; v=0 or 1; w=1 or 0; and x=1-16. 2.-3. (canceled)
 4. Therapeuticmolecule of claim 1, wherein the first ligand A1 or B1 comprises orconsists of an immunoglobulin, a binding domain of an immunoglobulin ora binding fragment of an immunoglobulin, such as an antibody, an IgG, amolecule comprising or consisting of a Vhh domain or Vh domain, a Fab,an scFv, an Fv, a dAb, an F(ab)₂, Fcab fragment, or comprise(s) orconsist(s) of at least one non-proteinaceous ligand and/or at least oneproteinaceous ligand, the ligand for binding to a cell-surface moleculesuch as EGF or a cytokine, with the proviso that the first ligand andthe second ligand are the same or are different.
 5. Therapeutic moleculeof claim 1, wherein the first ligand A1 or B1 binds to a tumor-cellepitope, preferably a tumor-cell specific epitope, of a tumor-cellreceptor, preferably a tumor-cell specific receptor, preferably selectedfrom CD71, CA125, EpCAM(17-1A), CD52, CEA, CD44v6, FAP, EGF-IR,integrin, syndecan-1, vascular integrin alpha-V beta-3, HER2, EGFR,CD20, CD22, Folate receptor 1, CD146, CD56, CD19, CD138, CD27L receptor,PSMA, CanAg, integrin-alphaV, CA6, CD33, mesothelin, Cripto, CD3, CD30,CD239, CD70, CD123, CD352, DLL3, CD25, ephrinA4, MUC1, Trop2, CEACAM5,CEACAM6, HER3, CD74, PTK7, Notch3, FGF2, C4.4A, FLT3, CD38, FGFR3, CD7,PD-L1, CTLA4, CD52, PDGFRA, VEGFR1, VEGFR2, more preferably selectedfrom CD71, EGFR and HER2, with the proviso that the first ligand and thesecond ligand bind to the same or to a different tumor-cell epitope,preferably a tumor-cell specific epitope, and/or wherein the tumor-cellreceptor, preferably the tumor-cell specific receptor, to which thefirst ligand can bind is the same as, or is different from thetumor-cell receptor, preferably the tumor-cell specific receptor, towhich the second ligand can bind.
 6. Therapeutic molecule of claim 1,wherein the first ligand A1 or B1 comprises or consists of cetuximab,daratumumab, gemtuzumab, trastuzumab, panitumumab, brentuximab,inotuzumab, moxetumomab, polatuzumab, obinutuzumab, OKT-9 anti-CD71monoclonal antibody of the IgG type, pertuzumab, rituximab, ofatumumab,Herceptin, alemtuzumab, pinatuzumab, OKT-10 anti-CD38 monoclonalantibody, an antibody of Table A2 or Table A3 or Table A4, preferablycetuximab or trastuzumab or OKT-9, or at least one tumor-cell receptorbinding-domain thereof and/or at least one tumor-cell receptorbinding-fragment thereof which are preferably (a) tumor-cell specificreceptor binding-domain(s) and/or (a) tumor-cell specific receptorbinding-fragment(s), with the proviso that the first ligand is the sameor different from the second ligand. 7.-8. (canceled)
 9. Therapeuticmolecule of claim 1, wherein the first effector moiety A1 or B1comprises or consist of at least one of any one or more of anoligonucleotide, a nucleic acid and a xeno nucleic acid, preferablyselected from any one or more of a vector, a gene, a cell suicideinducing transgene, deoxyribonucleic acid (DNA), ribonucleic acid (RNA),anti-sense oligonucleotide (ASO, AON), short interfering RNA (siRNA),microRNA (miRNA), DNA aptamer, RNA aptamer, mRNA, mini-circle DNA,peptide nucleic acid (PNA), phosphoramidate morpholino oligomer (PMO),locked nucleic acid (LNA), bridged nucleic acid (BNA),2′-deoxy-2′-fluoroarabino nucleic acid (FANA), 2′-O-methoxyethyl-RNA(MOE), 2′-0,4′-aminoethylene bridged nucleic acid, 3′-fluoro hexitolnucleic acid (FHNA), a plasmid, glycol nucleic acid (GNA) and threosenucleic acid (TNA), or a derivative thereof, more preferably a BNA, forexample a BNA for silencing HSP27 protein expression, with the provisothat the first effector moiety and the second effector moiety are thesame or are different.
 10. Therapeutic molecule of claim 1, wherein thefirst effector moiety A1 or B1 comprises or consists of at least oneproteinaceous molecule, preferably selected from any one or more of apeptide, a protein, an enzyme such as urease and Cre-recombinase, aproteinaceous toxin, a ribosome-inactivating protein, at least oneprotein toxin selected from Table A5 and/or a bacterial toxin, a planttoxin, more preferably selected from any one or more of a viral toxinsuch as apoptin; a bacterial toxin such as Shiga toxin, Shiga-liketoxin, Pseudomonas aeruginosa exotoxin (PE) or exotoxin A of PE,full-length or truncated diphtheria toxin (DT), cholera toxin; a fungaltoxin such as alpha-sarcin; a plant toxin includingribosome-inactivating proteins and the A chain of type 2ribosome-inactivating proteins such as dianthin e.g. dianthin-30 ordianthin-32, saporin e.g. saporin-S3 or saporin-S6, bouganin orde-immunized derivative debouganin of bouganin, shiga-like toxin A,pokeweed antiviral protein, ricin, ricin A chain, modeccin, modeccin Achain, abrin, abrin A chain, volkensin, volkensin A chain, viscumin,viscumin A chain; or an animal or human toxin such as frog RNase, orgranzyme B or angiogenin from humans, or any fragment or derivativethereof; preferably the protein toxin is dianthin and/or saporin, withthe proviso that the first effector moiety/moieties and the secondeffector moiety/moieties are the same or are different.
 11. (canceled)12. Therapeutic molecule of claim 1, wherein the therapeutic moleculecomprises or consists of any one of Gemtuzumab ozogamicin, Brentuximabvedotin, Trastuzumab emtansine, Inotuzumab ozogamicin, Moxetumomabpasudotox and Polatuzumab vedotin and an antibody-drug conjugate ofTable A2 and Table A3, or at least one tumor-cell specific receptorbinding-domain thereof and/or at least one tumor-cell specific receptorbinding-fragment thereof which are preferably (a) tumor-cell specificreceptor binding-domain(s) and/or (a) tumor-cell specific receptorbinding-fragment(s), with the proviso that the therapeutic molecule andthe second therapeutic molecule are the same or are different. 13.Therapeutic molecule of claim 1, wherein the saponin C is a triterpenoidsaponin or a bisdesmosidic triterpene saponin, belonging to the type ofa 12,13-dehydrooleanane with an aldehyde function in position C-23 andoptionally comprising a glucuronic acid function in a carbohydratesubstituent at the C-3beta-OH group of the saponin, and/or a saponinisolated from a Gypsophila species and/or a Saponaria species and/or anAgrostemma species and/or a Quillaja species such as Quillaja saponaria-or, wherein the saponin C is a single specific saponin or is a mixtureof two or more different saponins, such as one or more of the saponinsin Table A1 or Scheme I, SO1861, SA1657, GE1741, SA1641, QS-21, QS-21A,QS-21 A-api, QS-21 A-xyl, QS-21B, QS-21 B-api, QS-21 B-xyl, QS-7-xyl,QS-7-api, QS-17-api, QS-17-xyl, QS1861, QS1862, Quillajasaponin,Saponinum album, QS-18, Quil-A, Gyp1, gypsoside A, AG1, AG2, 501542,501584, 501658, 501674, 501832, or any of their stereomers and/or anycombinations thereof, preferably the saponin is SO1861 and/or GE1741and/or SA1641 and/or QS-21 and/or saponin with a quillaic acid aglyconcore, a Gal-(1→2)-[Xyl-(1→3)]-GlcA carbohydrate substituent at theC-3beta-OH group and aGlc-(1→3)-Xyl-(1→4)-Rha-(1→2)-[Xyl-(1→3)-4-OAc-Qui-(1→4)]-Fuccarbohydrate substituent at the C-28-OH group, and/or is3-O-beta-D-galactopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)]-beta-D-glucuronopyranosylquillaic acid28-O-beta-D-glucopyranosyl-(1→3)-beta-D-xylopyranosyl-(1→4)-alpha-L-rhamnopyranosyl-(1→2)-[beta-D-xylopyranosyl-(1→3)-4-OAc-beta-D-quinovopyranosyl-(1→4)]-beta-D-fucopyranoside,more preferably the saponin is SO1861 and/or QS-21 wherein the saponin Cis a bisdesmosidic saponin having a molecular mass of at least 1.500Dalton and comprising an oleanan-type triterpene containing an aldehydegroup at the C-23 position and optionally a hydroxyl group at the C-16position, with a first branched carbohydrate side chain at the C-3position which first branched carbohydrate side chain optionallycontains glucuronic acid, wherein the saponin contains an ester groupwith a second branched carbohydrate side chain at the C-28 positionwhich second branched carbohydrate chain preferably comprises at leastfour carbohydrate units, optionally containing at least one acetylresidue such as two acetyl residues and/or at least one deoxycarbohydrates and/or a quinovose and/or a glucose and/or4-methoxycinnamic acid and/or optionally comprising5-O-[5-O-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoicacid and/or optionally comprising5-O[5-O-Rha-(1→2)-Ara/Api-3,5-dihydroxy-6-methyl-octanoyl]-3,5-dihydroxy-6-methyl-octanoicacid bound to a carbohydrate via an ester bond, or wherein the at leastone saponin is QS-21 or any one or more of QS-21A, QS-21 A-api, QS-21A-xyl, QS-21B, QS-21 B-api, QS-21 B-xyl, QS-7-xyl, QS-7-api, QS-17-api,QS-17-xyl, QS-18, QS1861, protonated QS1861 (QS1862), Quil-A, or whereinthe saponin C is a bisdesmosidic triterpene saponin belonging to thetype of a 12,13-dehydrooleanane with an aldehyde function in positionC-23, wherein the saponin C is covalently coupled to an amino-acidresidue of the first ligand A1 or B1 and/or the first effector moiety B1or A1 and/or the second ligand A2 or B2 and/or the second effectormoiety B2 or A2 via the aldehyde function in the saponin C, preferablysaid aldehyde function in position C-23, preferably via a linker L2, L4,L6, L8, and/or L9 and/or L10, more preferably via a cleavable linker L2,L4, L6, L8, and/or L9 and/or L10, wherein the amino-acid residuepreferably is selected from cysteine and lysine. 14.-19. (canceled) 20.Therapeutic molecule of claim 1, wherein the first ligand A1 or B1and/or the first effector moiety B1 or A1 comprise(s) one or more thanone covalently bound saponin C, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32,64, 128 or 1-100 saponins, or any number of saponins therein between,such as 7, 9, 12 saponins.
 21. Therapeutic molecule of claim 1, whereinthe first ligand A1 or B1 and/or the first effector moiety B1 or A1comprise(s) one or more than one covalently bound saponin C, wherein thesaponin(s) C is/are covalently bound directly to an amino-acid residueof the first ligand A1 or B1 and/or the first effector moiety B1 or A1when r, s and v are 0, preferably to a cysteine and/or to a lysine,and/or is/are covalently bound via at least one linker L2, L4 and/or L9,or via at least one cleavable linker L2, L4 and/or L9 and/or via atleast one oligomeric or polymeric scaffold L3, preferably 1-8 of suchscaffolds or 2-4 of such scaffolds, wherein the at least one scaffold isoptionally based on a dendron, wherein 1-32 saponins, preferably 2, 3,4, 5, 6, 8, 10, 16, 32 saponins, or any number of saponins thereinbetween, such as 7, 9, 12 saponins, are covalently bound to the at leastone scaffold.
 22. Therapeutic molecule of claim 1, wherein the saponin Cis a bisdesmosidic triterpene saponin belonging to the type of a12,13-dehydrooleanane with an aldehyde function in position C-23,wherein the saponin C is covalently coupled to an amino-acid residue ofthe first ligand A1 or B1 and/or the first effector moiety B1 or A1 viathe aldehyde function in the saponin C, preferably said aldehydefunction in position C-23, via a cleavable linker L2, L4 and/or L9,wherein the amino-acid residue preferably is selected from cysteine andlysine, and wherein the cleavable linker L2, L4 and/or L9 is subject tocleavage under acidic conditions, reductive conditions, enzymaticconditions or light-induced conditions, and preferably the cleavablelinker comprises a hydrazone bond or a hydrazide bond subject tocleavage under acidic conditions when bound to saponin, and/or comprisesa bond susceptible to proteolysis, for example proteolysis by CathepsinB, when bound to saponin, and/or the cleavable linker comprises adisulphide bond susceptible to cleavage under reductive conditions. 23.Therapeutic molecule of claim 1, wherein the saponin C is abisdesmosidic triterpene saponin belonging to the type of a12,13-dehydrooleanane with an aldehyde function in position C-23,wherein the saponin C is covalently coupled to an amino-acid residue ofthe first ligand A1 or B1 and/or the first effector moiety B1 or A1 viathe aldehyde function in the saponin C, preferably said aldehydefunction in position C-23, via a cleavable linker L2, L4 and/or L9,wherein the amino-acid residue preferably is selected from cysteine andlysine, and wherein the cleavable linker L2, L4 and/or L9 is subject tocleavage in vivo under acidic conditions as present in endosomes and/orlysosomes of mammalian cells, preferably human cells, preferably at pH4.0-6.5, and more preferably at pH≤5.5.
 24. Therapeutic molecule ofclaim 1, wherein the polymeric or oligomeric scaffold L3 comprises apolymeric or oligomeric structure and comprises a chemical group, thechemical group for covalently coupling of the polymeric or oligomericscaffold L3 to the amino-acid residue of the first ligand and/or thefirst effector moiety and/or the second ligand and/or the secondeffector moiety. 25.-28. (canceled)
 29. Therapeutic molecule of claim 1,wherein the at least one saponin is covalently bound to the first ligandand/or to the first effector moiety via at least one linker comprising atri-functional linker L9 when j=1, to which tri-functional linker boththe first ligand and the at least one first effector moiety are bound,preferably the tri-functional linker is the trifunctional linker ofScheme II.
 30. Therapeutic molecule of claim 1, wherein the first ligandA1 or B1 and/or the first effector moiety B1 or A1 comprise(s) one ormore than one covalently bound saponin C, wherein the saponin(s) Cis/are covalently bound via at least one oligomeric or polymericscaffold L3, preferably 1-8 of such scaffolds or 2-4 of such scaffolds,wherein 1-32 saponins, preferably 2, 3, 4, 5, 6, 8, 10, 16, 32 saponins,or any number of saponins therein between, such as 7, 9, 12 saponins,are covalently bound to the at least one scaffold, and wherein thepolymeric or oligomeric structure of the scaffold L3 comprises a linear,branched and/or cyclic polymer, oligomer, dendrimer, dendron,dendronized polymer, dendronized oligomer, a DNA, a polypeptide,poly-lysine, a poly-ethylene glycol, or an assembly of these polymericor oligomeric structures which assembly is preferably built up bycovalent cross-linking.
 31. Therapeutic molecule of claim 1, wherein thefirst ligand A1 or B1 and/or the first effector moiety B1 or A1comprise(s) one or more than one covalently bound saponin C, wherein thesaponin(s) C is/are covalently bound directly to an amino-acid residueof the first ligand A1 or B1 and/or the first effector moiety B1 or A1when r, s and v are 0, preferably to a cysteine and/or to a lysine,and/or is/are covalently bound via at least one linker L2, L4 and/or L9,or via at least one cleavable linker L2, L4 and/or L9 and/or via atleast one oligomeric or polymeric scaffold L3, preferably 1-8 of suchscaffolds or 2-4 of such scaffolds, wherein the at least one scaffold isoptionally based on a dendron, wherein 1-32 saponins, preferably 2, 3,4, 5, 6, 8, 10, 16, 32 saponins, or any number of saponins thereinbetween, such as 7, 9, 12 saponins, are covalently bound to the at leastone scaffold, and wherein the first ligand A1 or B1 is covalently boundto the first effector moiety B1 or A1, respectively, via at least onelinker L1.
 32. (canceled)
 33. Therapeutic molecule of claim 1, whereinr=0, s=0, v=0, s=0, v=0, p=0, t=0, ligand A1 is a monoclonal antibody orat least one binding fragment or -domain thereof selected from animmunoglobulin, a binding domain of an immunoglobulin or a bindingfragment of an immunoglobulin, such as an antibody, an IgG, a moleculecomprising or consisting of a Vhh domain or Vh domain, a Fab, an scFv,an Fv, a dAb, an F(ab)₂, Fcab fragment, m=1, effector moiety B1 is atleast one of an oligonucleotide, a nucleic acid and a xeno nucleic acid,preferably selected from any one or more of a vector, a gene, a cellsuicide inducing transgene, deoxyribonucleic acid (DNA), ribonucleicacid (RNA), anti-sense oligonucleotide (ASO, AON), short interfering RNA(siRNA), microRNA (miRNA), DNA aptamer, RNA aptamer, mRNA, mini-circleDNA, peptide nucleic acid (PNA), phosphoramidate morpholino oligomer(PMO), locked nucleic acid (LNA), bridged nucleic acid (BNA),2′-deoxy-2′-fluoroarabino nucleic acid (FANA), 2′-O-methoxyethyl-RNA(MOE), 2′-0,4′-aminoethylene bridged nucleic acid, 3′-fluoro hexitolnucleic acid (FHNA), a plasmid, glycol nucleic acid (GNA) and threosenucleic acid (TNA), or a derivative thereof, preferably a BNA, eitherq=0, n=1 and u=2-4, or q=1, n=2-4, u=2-4 and linker L1 is at least oneoligomeric or polymeric scaffold L3, preferably 1-8 of such scaffolds or2-4 of such scaffolds, wherein the at least one scaffold is optionallybased on a dendron, wherein 1-32 saponins, preferably 2, 3, 4, 5, 6, 8,10, 16, 32 saponins, or any number of saponins therein between, such as7, 9, 12 saponins, are covalently bound to the at least one scaffold.34. (canceled)
 35. Pharmaceutical composition comprising the therapeuticmolecule with chemical structure of COMPOUND I according to claim 1, andoptionally further comprising a pharmaceutically acceptable excipient.36.-37. (canceled)
 38. Method for the treatment or prophylaxis of cancerin a patient in need thereof comprising administering to the patient thepharmaceutical composition of claim 35, wherein the ligand A1 or B1 canbind to a tumor-cell epitope, preferably a tumor-cell specific epitope,on a tumor-cell surface molecule, preferably a tumor cell-specificsurface molecule. 39.-42. (canceled)